Methods for detecting target analytes and enzymatic reactions

ABSTRACT

A microsphere-based analytic chemistry system and method for making the same is disclosed in which microspheres or particles carrying bioactive agents may be combined randomly or in ordered fashion and dispersed on a substrate to form an array while maintaining the ability to identify the location of bioactive agents and particles within the array using an optically interrogatable, optical signature encoding scheme. A wide variety of modified substrates may be employed which provide either discrete or non-discrete sites for accommodating the microspheres in either random or patterned distributions. The substrates may be constructed from a variety of materials to form either two-dimensional or three-dimensional configurations. In a preferred embodiment, a modified fiber optic bundle or array is employed as a substrate to produce a high density array. The disclosed system and method have utility for detecting target analytes and screening large libraries of bioactive agents.

This application is a continuation-in-part of U.S. Ser. No. 09/786,896,filed Sep. 10, 1999, now pending, which is a national phase applicationof PCT application US 99/20914, which claims priority of U.S. Ser. No.09/151,877, filed Sep. 11, 1998, now U.S. Pat. No. 6,327,410, issuedDec. 4, 2001, which is a continuation-in-part of U.S. Ser. No.08/818,199, filed Mar. 14, 1997, now U.S. Pat. No. 6,023,540, issuedFeb. 8, 2000. This application is also a continuation-in-part of U.S.Ser. No. 09/840,012, filed Apr. 20, 2001, now pending, which is acontinuation of U.S. Ser. No. 09/450,829, filed Nov. 29, 1999, now U.S.Pat. No. 6,266,459, issued Jul. 24, 2001, which is a continuation ofU.S. Ser. No. 08/818,199, filed Mar. 14, 1997, now U.S. Pat. No.6,023,540, issued Feb. 8, 2000. This application is also acontinuation-in-part of U.S. Ser. No. 09/925,292, filed Aug. 8, 2001,now pending, which is a continuation of U.S. Ser. No. 09/151,877, filedSep. 11, 1998, now U.S. Pat. No. 6,327,410, issued Dec. 4, 2001, whichis a continuation-in-part of U.S. Ser. No. 08/818,199, filed Mar. 14,1997, now U.S. Pat. No. 6,023,540, issued Feb. 8, 2000. This applicationis also a continuation-in-part of U.S. Ser. No. 09/816,651, filed Mar.23, 2001, now pending, which is a continuation of U.S. Ser. No.09/450,829, filed Nov. 29, 1999, now U.S. Pat. No. 6,266,459, issuedJul. 24, 2001, which is a continuation of U.S. Ser. No. 08/818,199,filed Mar. 14, 1997, now U.S. Pat. No. 6,023,540, issued Feb. 8, 2000.All of the above patent applications are expressly incorporated hereinby reference.

BACKGROUND OF THE INVENTION

The use of optical fibers and optical fiber strands in combination withlight absorbing dyes for chemical analytical determinations hasundergone rapid development, particularly within the last decade. Theuse of optical fibers for such purposes and techniques is described byMilanovich et al., “Novel Optical Fiber Techniques For MedicalApplication”, Proceedings of the SPIE 28th Annual InternationalTechnical Symposium On Optics and Electro-Optics, Volume 494, 1980;Seitz, W. R., “Chemical Sensors Based On Immobilized Indicators andFiber Optics” in C.R.C. Critical Reviews In Analytical Chemistry, Vol.19,1988, pp. 135-173; Wolfbeis, O. S., “Fiber Optical Fluorosensors InAnalytical Chemistry” in Molecular Luminescence Spectroscopy, Methodsand Applications (S. G. Schulman, editor), Wiley & Sons, New York(1988); Angel, S. M., Spectroscopy 2 (4):38 (1987); Walt, et al.,“Chemical Sensors and Microinstrumentation”, ACS Symposium Series, Vol.403,1989, p. 252, and Wolfbeis, O. S., Fiber Optic Chemical Sensors, Ed.CRC Press, Boca Raton, Fla., 1991, 2nd Volume.

When using an optical fiber in an in vitro/in vivo sensor, one or morelight absorbing dyes are located near its distal end. Typically, lightfrom an appropriate source is used to illuminate the dyes through thefiber's proximal end. The light propagates along the length of theoptical fiber; and a portion of this propagated light exits the distalend and is absorbed by the dyes. The light absorbing dye may or may notbe immobilized; may or may not be directly attached to the optical fiberitself; may or may not be suspended in a fluid sample containing one ormore analytes of interest; and may or may not be retainable forsubsequent use in a second optical determination.

Once the light has been absorbed by the dye, some light of varyingwavelength and intensity returns, conveyed through either the same fiberor collection fiber(s) to a detection system where it is observed andmeasured. The interactions between the light conveyed by the opticalfiber and the properties of the light absorbing dye provide an opticalbasis for both qualitative and quantitative determinations.

Of the many different classes of light absorbing dyes whichconventionally are employed with bundles of fiber strands and opticalfibers for different analytical purposes are those more commoncompositions that emit light after absorption termed “fluorophores” andthose which absorb light and internally convert the absorbed light toheat, rather than emit it as light, termed “chromophores.”

Fluorescence is a physical phenomenon based upon the ability of somemolecules to absorb light (photons) at specified wavelengths and thenemit light of a longer wavelength and at a lower energy. Substances ableto fluoresce share a number of common characteristics: the ability toabsorb light energy at one wavelength λ_(ab); reach an excited energystate; and subsequently emit light at another light wavelength, λ_(em).The absorption and fluorescence emission spectra are individual for eachfluorophore and are often graphically represented as two separate curvesthat are slightly overlapping. The same fluorescence emission spectrumis generally observed irrespective of the wavelength of the excitinglight and, accordingly, the wavelength and energy of the exciting lightmay be varied within limits; but the light emitted by the fluorophorewill always provide the same emission spectrum. Finally, the strength ofthe fluorescence signal may be measured as the quantum yield of lightemitted. The fluorescence quantum yield is the ratio of the number ofphotons emitted in comparison to the number of photons initiallyabsorbed by the fluorophore. For more detailed information regardingeach of these characteristics, the following references are recommended:Lakowicz, J. R., Principles of Fluorescence Spectroscopy, Plenum Press,New York, 1983; Freifelder, D., Physical Biochemistry, second edition,W. H. Freeman and Company, New York, 1982; “Molecular LuminescenceSpectroscopy Methods and Applications: Part I” (S. G. Schulman, editor)in Chemical Analysis, vol. 77, Wiley & Sons, Inc., 1985; The Theory ofLuminescence, Stepanov and Gribkovskii, Iliffe Books, Ltd., London,1968.

In comparison, substances which absorb light and do not fluoresceusually convert the light into heat or kinetic energy. The ability tointernally convert the absorbed light identifies the dye as a“chromophore.” Dyes which absorb light energy as chromophores do so atindividual wavelengths of energy and are characterized by a distinctivemolar absorption coefficient at that wavelength. Chemical analysisemploying fiber optic strands and absorption spectroscopy using visibleand ultraviolet light wavelengths in combination with the absorptioncoefficient allow for the determination of concentration for specificanalyses of interest by spectral measurement. The most common use ofabsorbance measurement via optical fibers is to determine concentrationwhich is calculated in accordance with Beers' law; accordingly, at asingle absorbance wavelength, the greater the quantity of thecomposition which absorbs light energy at a given wavelength, thegreater the optical density for the sample. In this way, the totalquantity of light absorbed directly correlates with the quantity of thecomposition in the sample.

Many of the recent improvements employing optical fiber sensors in bothqualitative and quantitative analytical determinations concern thedesirability of depositing and/or immobilizing various light absorbingdyes at the distal end of the optical fiber. In this manner, a varietyof different optical fiber chemical sensors and methods have beenreported for specific analytical determinations and applications such aspH measurement, oxygen detection, and carbon dioxide analyses. Thesedevelopments are exemplified by the following publications: Freeman, etal., Anal Chem. 53:98 (1983); Lippitsch et al., Anal. Chem. Acta. 205:1,(1988); Wolfbeis et al., Anal. Chem. 60:2028 (1988); Jordan, et al.,Anal. Chem. 59:437 (1987); Lubbers et al., Sens. Actuators 1983;Munkholm et al., Talanta 35:109 (1988); Munkholm et al., Anal. Chem.58:1427 (1986); Seitz, W. R., Anal. Chem. 56:16 A-34A (1984); Peterson,et al., Anal. Chem. 52:864 (1980): Saari, et al., Anal. Chem. 54:821(1982); Saari, et al., Anal. Chem. 55:667 (1983); Zhujun et al., Anal.Chem. Acta. 160:47 (1984); Schwab, et al., Anal. Chem. 56:2199 (1984);Wolfbeis, O. S., “Fiber Optic Chemical Sensors”, Ed. CRC Press, BocaRaton, Fla., 1991, 2nd Volume; and Pantano, P., Walt, D. R., Anal.Chem., 481A-487A, Vol. 67, (1995).

More recently, fiber optic sensors have been constructed that permit theuse of multiple dyes with a single, discrete fiber optic bundle. U.S.Pat. Nos. 5,244,636 and 5,250,264 to Walt, et al. disclose systems foraffixing multiple, different dyes on the distal end of the bundle, theteachings of each of these patents being incorporated herein by thisreference. The disclosed configurations enable separate optical fibersof the bundle to optically access individual dyes. This avoids theproblem of deconvolving the separate signals in the returning light fromeach dye, which arises when the signals from two or more dyes arecombined, each dye being sensitive to a different analyte, and there issignificant overlap in the dyes' emission spectra.

In the two previous patents multiple chemical functionalities wereplaced at the end of a single optical fiber bundle sensor. Thisconfiguration yielded an analytic chemistry sensor that could beremotely monitored via the typically small bundle. A potential drawback,however, was the difficulty in applying the various chemistriesassociated with the chemical functionalities; the functionalities werebuilt on the sensor in a serial fashion. Accordingly, compositions andmethods are desirable that allow the generation of large arrays that canbe either encoded or decoded to allow the detection of target analytes.The arrays can be fiber optic arrays or arrays on other array substratesand can include microspheres.

SUMMARY OF THE INVENTION

Accordingly, in one aspect the invention provides a method of detectinga target analyte in a sample comprising providing an array comprising anarray substrate, wherein the array substrate is other than a fiber opticbundle, and at least first and second sites wherein first and secondreaction components are immobilized at said first and second sites,respectively, contacting said array substrate with the sample anddetecting a change in an optical property around at least the first siteas an indication of the interaction between the target analyte and atleast the first reaction component.

In addition the invention provides a method of detecting a targetanalyte in a sample comprising providing an array comprising an arraysubstrate comprising discrete sites and a population of microspherescomprising at least first and second subpopulations comprising first andsecond reaction components respectively and a detection molecule. Themethod further includes contacting the array with the sample anddetecting a change in an optical property around at least the firstmicrosphere as an indication of the interaction between the targetanalyte and at least the first reaction component.

The invention also provides a method of detecting an enzymatic reactioncomprising providing an array comprising an array substrate comprisingdiscrete sites, and a population of microspheres randomly distributed onthe array substrate, wherein the microspheres comprise at least oneenzyme, contacting the array with a sample comprising a target analyte,wherein the target analyte is an enzyme substrate, monitoring a signalin a region surrounding the microspheres, whereby detection of thesignal provides an indication of the reaction between the enzyme and theenzyme substrate.

In addition the invention provides a method of detecting an enzymaticreaction comprising providing an array comprising an array substratecomprising discrete sites and a population of microspheres randomlydistributed on said array substrate, the population comprising first andsecond subpopulations, wherein the first and second subpopulationscomprise first and second discrete oligonucleotides, respectively,attached to the microspheres, contacting the array with a compositioncomprising an enzyme, monitoring a signal in a region surrounding themicrospheres, whereby detection of the signal provides an indication ofthe reaction between the enzyme and at least one of the discreteoligonucleotides.

BRIEF DESCRIPTION OF THE DRAWINGS

In the accompanying drawings, reference characters refer to the sameparts throughout the different views. The drawings are not necessarilyto scale; emphasis has instead been placed upon illustrating theprinciples of the invention. Of the drawings:

FIG. 1 is a schematic diagram illustrating the optical signatureencoding and chemical functionalizing of the microspheres according tothe present invention;

FIG. 2 is a process diagram describing the preparation, encoding, andfunctionalizing of the microspheres of the present invention;

FIG. 3 is a schematic diagram illustrating a microsphere systemincluding microspheres with different chemical functionalities andencoded descriptions of the functionalities;

FIG. 4 is a schematic diagram of the inventive fiber optic sensor andassociated instrumentation and control system;

FIGS. 5A and 5B are micrographs illustrating the preferred technique forattaching or affixing the microspheres to the distal end of the opticalfiber bundle;

FIG. 6 is a process diagram describing well formation in the opticalfiber bundle and affixation of the microspheres in the wells;

FIGS. 7A and 7B are micrographs showing the array of microspheres intheir corresponding wells prior and subsequent to physical agitation,tapping and air pulsing, demonstrating the electrostatic binding of themicrospheres in the wells;

FIGS. 8A, 8B, and 8C are micrographs from alkaline phosphatasemicrospheres when exposed to fluorescein diphosphate, at the fluoresceinemission wavelength, at an encoding wavelength for DilC, and at anencoding wavelength for TRC, respectively;

FIGS. 9A and 9B are micrographs showing the optical signal fromβ-galactosidase microspheres when exposed to fluoresceinβ-galactopyranoside at the fluorescein emission wavelength and at anencoding wavelength for DilC, respectively; and

FIGS. 10A and 10B are micrographs showing the optical response fromrabbit antibody microspheres prior to and post, respectively, exposureto fluorescein labeled antigens.

FIGS. 11A and 11B are micrographs depicting the optical response frombeads synthesized with DNA on the bead surface, following a 10 min.hybridization with a Cy3-labeled probe complementary to the sequence ofthe DNA immobilized on the bead. Beads were randomly distributed on A)an etched optical imaging fiber or B) a patterned polymer (polyurethane)substrate (a chip). Following hybridization with 5 nM Cy3-labeled probe,the substrates were placed in buffer for optical readout on an imagingsystem. A) was imaged through the proximal end, with the distal (beaded)end in buffer solution. B) was imaged directly from the top, through acoverslip.

FIGS. 12A, 12B and 12C are micrographs depicting the optical responsesbetween different substrates. The substrate in A) and B) is an etchedoptical imaging fiber, and the substrate in C) is a chip. Data wereobtained as described in FIG. 11, and quantified to determine meanintensity and variability.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is based on the combination of high-density arraysand a novel detection mechanism wherein labels or signals are detectedon an array in a region surrounding a site on the array. That is,previously, detection on arrays involved the detection of targets whilethe targets were attached to the array or array substrate. However, thepresent invention provides methods and compositions for monitoring thepresence of a target analyte by detecting a change in an opticalproperty around at least a first discrete site on an array.

In one embodiment, the present invention is based on two synergisticinventions: 1) the development of a bead-based analytic chemistry systemin which beads, also termed microspheres, carrying different chemicalfunctionalities may be mixed together while the ability is retained toidentify the functionality of each bead using an opticallyinterrogatable encoding scheme (an “optical signature”); and 2) the useof a substrate comprising a patterned surface containing individualsites that can bind or associate individual beads. In preferredembodiments the substrate is patterned. This allows the synthesis of thebioactive agents (i.e. compounds such as nucleic acids and antibodies)to be separated from their placement on an array, i.e. the bioactiveagents may be synthesized on the beads, and then the beads are randomlydistributed on a patterned surface. Since the beads are first coded withan optical signature, this means that the array can later be “decoded”,i.e. after the array is made, a correlation of the location of anindividual site on the array with the bead or bioactive agent at thatparticular site can be made. This means that the beads may be randomlydistributed on the array, a fast and inexpensive process as compared toeither the in situ synthesis or spotting techniques of the prior art.Once the array is loaded with the beads, the array can be decoded, orcan be used, with full or partial decoding occurring after testing, asis more fully outlined below.

Accordingly, the present invention provides array compositionscomprising at least a first substrate with a surface comprisingindividual sites. By “array” herein is meant a plurality of bioactiveagents in an array format; the size of the array will depend on thecomposition and end use of the array. Arrays containing from about 2different bioactive agents (i.e. different beads) to many millions canbe made, with very large fiber optic arrays being possible. Generally,the array will comprise from two to as many as a billion or more,depending on the size of the beads and the substrate, as well as the enduse of the array, thus very high density, high density, moderatedensity, low density and very low density arrays may be made. Preferredranges for very high density arrays are from about 10,000,000 to about2,000,000,000, with from about 100,000,000 to about 1,000,000,000 beingpreferred. High density arrays range about 100,000 to about 10,000,000,with from about 1,000,000 to about 5,000,000 being particularlypreferred. Moderate density arrays range from about 10,000 to about50,000 being particularly preferred, and from about 20,000 to about30,000 being especially preferred. Low density arrays are generally lessthan 10,000, with from about 1,000 to about 5,000 being preferred. Verylow density arrays are less than 1,000, with from about 10 to about 1000being preferred, and from about 100 to about 500 being particularlypreferred. In some embodiments, the compositions of the invention maynot be in array format; that is, for some embodiments, compositionscomprising a single bioactive agent may be made as well. In addition, insome arrays, multiple substrates may be used, either of different oridentical compositions. Thus for example, large arrays may comprise aplurality of smaller substrates.

In addition, one advantage of the present compositions is thatparticularly through the use of fiber optic technology, extremely highdensity arrays can be made. Thus for example, because beads of 200 nmcan be used, and very small fibers are known, it is possible to have asmany as 250,000 different fibers and beads in a 1 mm² fiber opticbundle, with densities of greater than 15,000,000 individual beads andfibers per 0.5 cm² obtainable.

The compositions comprise a substrate. By “substrate”, “array substrate”or “solid support” or other grammatical equivalents herein is meant anymaterial that can be modified to contain discrete individual sitesappropriate for the attachment or association of beads and is amenableto at least one detection method. As will be appreciated by those in theart, the number of possible substrates are very large, and include, butare not limited to, glass and modified or functionalized glass, plastics(including acrylics, polystyrene and copolymers of styrene and othermaterials, polypropylene, polyethylene, polybutylene, polyurethanes,TeflonJ, etc.), polysaccharides, nylon or nitrocellulose, compositematerials, ceramics, and plastic resins, silica or silica-basedmaterials including silicon and modified silicon, carbon, metals,inorganic glasses, plastics, optical fiber bundles, and a variety ofother polymers. In general, the substrates allow optical detection anddo not appreciably fluoresce.

In one embodiment, the substrate does not comprise the ends of anoptical fiber bundle.

In one embodiment, the substrate is planar, although as will beappreciated by those in the art, other configurations of substrates maybe used as well; for example, three dimensional configurations can beused, for example by embedding the beads in a porous block of plasticthat allows sample access to the beads and using a confocal microscopefor detection. Similarly, the beads may be placed on the inside surfaceof a tube, for flow-through sample analysis to minimize sample volume.Preferred substrates include optical fiber bundles as discussed below,and flat planar substrates such as glass, polystyrene and other plasticsand acrylics.

In one embodiment, at least one surface of the substrate is modified tocontain discrete, individual sites for later association ofmicrospheres. These sites may comprise physically altered sites, i.e.physical configurations such as wells or small depressions in thesubstrate that can retain the beads, such that a microsphere can rest inthe well, or the use of other forces (magnetic or compressive), orchemically altered or active sites, such as chemically functionalizedsites, electrostatically altered sites, hydrophobically/hydrophilicallyfunctionalized sites, spots of adhesive, etc.

The sites may be a pattern, i.e. a regular design or configuration, orrandomly distributed. A preferred embodiment utilizes a regular patternof sites such that the sites may be addressed in the X-Y coordinateplane. “Pattern” in this sense includes a repeating unit cell,preferably one that allows a high density of beads on the substrate.However, it should be noted that these sites may not be discrete sites.That is, it is possible to use a uniform surface of adhesive or chemicalfunctionalities, for example, that allows the attachment of beads at anyposition. That is, the surface of the substrate is modified to allowattachment of the microspheres at individual sites, whether or not thosesites are contiguous or non-contiguous with other sites. Thus, thesurface of the substrate may be modified such that discrete sites areformed that can only have a single associated bead, or alternatively,the surface of the substrate is modified and beads may go down anywhere,but they end up at discrete sites.

In one embodiment, the surface of the substrate is modified to containwells, i.e. depressions in the surface of the substrate. This may bedone as is generally known in the art using a variety of techniques,including, but not limited to, photolithography, stamping techniques,molding techniques and microetching techniques. As will be appreciatedby those in the art, the technique used will depend on the compositionand shape of the substrate.

In one embodiment, physical alterations are made in a surface of thesubstrate to produce the sites. In a preferred embodiment, the substrateis a fiber optic bundle and the surface of the substrate is a terminalend of the fiber bundle. In this embodiment, wells are made in aterminal or distal end of a fiber optic bundle comprising individualfibers. In this embodiment, the cores of the individual fibers areetched, with respect to the cladding, such that small wells ordepressions are formed at one end of the fibers. The required depth ofthe wells will depend on the size of the beads to be added to the wells.

In this embodiment, the microspheres suitably are non-covalentlyassociated in the wells, although the wells may additionally bechemically functionalized as is generally described below, cross-linkingagents may be used, or a physical barrier may be used, i.e. a film ormembrane over the beads.

In a further embodiment, the surface of the substrate is modified tocontain chemically modified sites, that can be used to attach, eithercovalently or non-covalently, the microspheres of the invention to thediscrete sites or locations on the substrate. “Chemically modifiedsites” in this context includes, but is not limited to, the addition ofa pattern of chemical functional groups including amino groups, carboxygroups, oxo groups and thiol groups, that can be used to covalentlyattach microspheres, which generally also contain corresponding reactivefunctional groups; the addition of a pattern of adhesive that can beused to bind the microspheres (either by prior chemicalfunctionalization for the addition of the adhesive or direct addition ofthe adhesive); the addition of a pattern of charged groups (similar tothe chemical functionalities) for the electrostatic attachment of themicrospheres, i.e. when the microspheres comprise charged groupsopposite to the sites; the addition of a pattern of chemical functionalgroups that renders the sites differentially hydrophobic or hydrophilic,such that the addition of similarly hydrophobic or hydrophilicmicrospheres under suitable experimental conditions will result inassociation of the microspheres to the sites on the basis ofhydroaffinity. For example, the use of hydrophobic sites withhydrophobic beads, in an aqueous system, drives the association of thebeads preferentially onto the sites. As outlined above, “pattern” inthis sense includes the use of a uniform treatment of the surface toallow attachment of the beads at discrete sites, as well as treatment ofthe surface resulting in discrete sites. As will be appreciated by thosein the art, this may be accomplished in a variety of ways.

The compositions of the invention further comprise a population ofmicrospheres. By “population” herein is meant a plurality of beads asoutlined above for arrays. Within the population are separatesubpopulations, which can be a single microsphere or multiple identicalmicrospheres. That is, in some embodiments, as is more fully outlinedbelow, the array may contain only a single bead for each bioactiveagent; preferred embodiments utilize a plurality of beads of each type.

By “microspheres” or “beads” or “particles” or grammatical equivalentsherein is meant small discrete particles. The composition of the beadswill vary, depending on the class of bioactive agent and the method ofsynthesis. Suitable bead compositions include those used in peptide,nucleic acid and organic moiety synthesis, including, but not limitedto, plastics, ceramics, glass, polystyrene, methylstyrene, acrylicpolymers, paramagnetic materials, thoria sol, carbon graphited, titaniumdioxide, latex or cross-linked dextrans such as Sepharose, cellulose,nylon, cross-linked micelles and teflon may all be used. “MicrosphereDetection Guide” from Bangs Laboratories, Fishers Ind. is a helpfulguide.

The beads need not be spherical; irregular particles may be used. Inaddition, the beads may be porous, thus increasing the surface area ofthe bead available for either bioactive agent attachment or tagattachment. The bead sizes range from nanometers, i.e. 100 nm, tomillimeters, i.e. 1 mm, with beads from about 0.2 micron to about 200microns being preferred, and from about 0.5 to about 5 micron beingparticularly preferred, although in some embodiments smaller beads maybe used.

FIG. 1 illustrates the construction of a bead or microsphere 10according to the principles of the present invention. In common with theprior art, the microsphere 10 is given a bioactive agent 12, which istypically applied to the microsphere's surface. The bioactive agent isdesigned so that in the presence of the analyte(s) to which it istargeted, an optical signature of the microsphere, possibly includingregion surrounding it, is changed.

It should be noted that a key component of the invention is the use of asubstrate/bead pairing that allows the association or attachment of thebeads at discrete sites on the surface of the substrate, such that thebeads do not move during the course of the assay.

In one embodiment each microsphere comprises two components: a bioactiveagent and an optical signature.

By “candidate bioactive agent” or “bioactive agent” or “chemicalfunctionality” or “binding ligand” herein is meant as used hereindescribes any molecule, e.g., protein, oligopeptide, small organicmolecule, polysaccharide, polynucleotide, etc. which can be attached tothe microspheres of the invention. It should be understood that thecompositions of the invention have two primary uses. In a preferredembodiment, as is more fully outlined below, the compositions are usedto detect the presence of a particular target analyte; for example, thepresence or absence of a particular nucleotide sequence or a particularprotein, such as an enzyme, an antibody or an antigen. In an alternatepreferred embodiment, the compositions are used to screen bioactiveagents, i.e. drug candidates, for binding to a particular targetanalyte.

Bioactive agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 100 and less than about 2,500 daltons.Bioactive agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The bioactiveagents often comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Bioactive agents are also found amongbiomolecules including peptides, nucleic acids, saccharides, fattyacids, steroids, purines, pyrimidines, derivatives, structural analogsor combinations thereof. Particularly preferred are nucleic acids andproteins.

Bioactive agents can be obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means. Knownpharmacological agents may be subjected to directed or random chemicalmodifications, such as acylation, alkylation, esterification and/oramidification to produce structural analogs.

In a preferred embodiment, the bioactive agents are proteins. By“protein” herein is meant at least two covalently attached amino acids,which includes proteins, polypeptides, oligopeptides and peptides. Theprotein may be made up of naturally occurring amino acids and peptidebonds, or synthetic peptidomimetic structures. Thus “amino acid”, or“peptide residue”, as used herein means both naturally occurring andsynthetic amino acids. For example, homo-phenylalanine, citrulline andnorleucine are considered amino acids for the purposes of the invention.The side chains may be in either the (R) or the (S) configuration. Inthe preferred embodiment, the amino acids are in the (S) orL-configuration. If non-naturally occurring side chains are used,non-amino acid substituents may be used, for example to prevent orretard in vivo degradations.

In one preferred embodiment, the bioactive agents are naturallyoccurring proteins or fragments of naturally occurring proteins. Thus,for example, cellular extracts containing proteins, or random ordirected digests of proteinaceous cellular extracts, may be used. Inthis way libraries of procaryotic and eukaryotic proteins may be madefor screening in the systems described herein. Particularly preferred inthis embodiment are libraries of bacterial, fungal, viral, and mammalianproteins, with the latter being preferred, and human proteins beingespecially preferred.

In a preferred embodiment, the bioactive agents are peptides of fromabout 5 to about 30 amino acids, with from about 5 to about 20 aminoacids being preferred, and from about 7 to about 15 being particularlypreferred. The peptides may be digests of naturally occurring proteinsas is outlined above, random peptides, or “biased” random peptides. By“randomized” or grammatical equivalents herein is meant that eachnucleic acid and peptide consists of essentially random nucleotides andamino acids, respectively. Since generally these random peptides (ornucleic acids, discussed below) are chemically synthesized, they mayincorporate any nucleotide or amino acid at any position. The syntheticprocess can be designed to generate randomized proteins or nucleicacids, to allow the formation of all or most of the possiblecombinations over the length of the sequence, thus forming a library ofrandomized bioactive proteinaceous agents.

In a preferred embodiment, a library of bioactive agents are used. Thelibrary should provide a sufficiently structurally diverse population ofbioactive agents to effect a probabilistically sufficient range ofbinding to target analytes. Accordingly, an interaction library must belarge enough so that at least one of its members will have a structurethat gives it affinity for the target analyte. Although it is difficultto gauge the required absolute size of an interaction library, natureprovides a hint with the immune response: a diversity of 10⁷-10⁸different antibodies provides at least one combination with sufficientaffinity to interact with most potential antigens faced by an organism.Published in vitro selection techniques have also shown that a librarysize of 10⁷ to 10⁸ is sufficient to find structures with affinity forthe target. Thus, in a preferred embodiment, at least 10⁶, preferably atleast 10⁷, more preferably at least 10⁸ and most preferably at least 10⁹different bioactive agents are simultaneously analyzed in the subjectmethods. Preferred methods maximize library size and diversity.

In one embodiment, the library is fully randomized, with no sequencepreferences or constants at any position. In a preferred embodiment, thelibrary is biased. That is, some positions within the sequence areeither held constant, or are selected from a limited number ofpossibilities. For example, in a preferred embodiment, the nucleotidesor amino acid residues are randomized within a defined class, forexample, of hydrophobic amino acids, hydrophilic residues, stericallybiased (either small or large) residues, towards the creation ofcysteines, for cross-linking, prolines for SH-3 domains, serines,threonines, tyrosines or histidines for phosphorylation sites, etc., orto purines, etc.

In a preferred embodiment, the bioactive agents are nucleic acids(generally called “probe nucleic acids” or “candidate probes” herein).By “nucleic acid” or “oligonucleotide” or grammatical equivalents hereinmeans at least two nucleotides covalently linked together. A nucleicacid of the present invention will generally contain phosphodiesterbonds, although in some cases, as outlined below, nucleic acid analogsare included that may have alternate backbones, comprising, for example,phosphoramide (Beaucage, et al., Tetrahedron, 49(10):1925 (1993) andreferences therein; Letsinger, J. Org. Chem., 35:3800 (1970); Sprinzl,et al., Eur. J. Biochem., 81:579 (1977); Letsinger, e t al., Nucl. AcidsRes., 14:3487 (1986); Sawai, et al., Chem. Lett., 805 (1984), Letsinger,et al., J. Am. Chem. Soc., 110:4470 (1988); and Pauwels, et al., ChemicaScripta, 26:141 (1986)), phosphorothioate (Mag, et al., Nucleic AcidsRes., 19:1437 (1991); and U.S. Pat. No. 5,644,048), phosphorodithioate(Briu, et al., J. Am. Chem. Soc., 111:2321 (1989)),O-methylphosphoroamidite linkages (see Eckstein, Oligonucleotides andAnalogues: A Practical Approach, Oxford University Press), and peptidenucleic acid backbones and linkages (see Egholm, J. Am. Chem. Soc.,114:1895 (1992); Meier, et al., Chem. Int. Ed. Engl., 31:1008 (1992);Nielsen, Nature, 365:566 (1993); Carlsson, et al., Nature, 380:207(1996), all of which are incorporated by reference)). Other analognucleic acids include those with positive backbones (Denpcy, et al.,Proc. Natl. Acad. Sci. USA, 92:6097 (1995)); non-ionic backbones (U.S.Pat. Nos. 5,386,023; 5,637,684; 5,602,240; 5,216,141; and 4,469,863;Kiedrowshi, et al., Angew. Chem. Intl. Ed. English, 30:423 (1991);Letsinger, et al., J. Am. Chem. Soc., 110:4470 (1988); Letsinger, etal., Nucleosides & Nucleotides, 13:1597 (1994); Chapters 2 and 3, ASCSymposium Series 580, “Carbohydrate Modifications in AntisenseResearch”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker, et al.,Bioorganic & Medicinal Chem. Lett., 4:395 (1994); Jeffs, et al., J.Biomolecular NMR, 34:17 (1994); Tetrahedron Lett., 37:743 (1996)) andnon-ribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook. Nucleic acids containing one or more carbocyclic sugarsare also included within the definition of nucleic acids (see Jenkins,et al., Chem. Soc. Rev., (1995) pp. 169-176). Several nucleic acidanalogs are described in Rawls, C & E News, Jun. 2, 1997, page 35. Allof these references are hereby expressly incorporated by reference.These modifications of the ribose-phosphate backbone may be done tofacilitate the addition of additional moieties such as labels, or toincrease the stability and half-life of such molecules in physiologicalenvironments. In addition, mixtures of naturally occurring nucleic acidsand analogs can be made. Alternatively, mixtures of different nucleicacid analogs, and mixtures of naturally occurring nucleic acids andanalogs may be made. The nucleic acids may be single stranded or doublestranded, as specified, or contain portions of both double stranded orsingle stranded sequence. The nucleic acid may be DNA, both genomic andcDNA, RNA or a hybrid, where the nucleic acid contains any combinationof deoxyribo- and ribo-nucleotides, and any combination of bases,including uracil, adenine, thymine, cytosine, guanine, inosine,xanthanine, hypoxanthanine, isocytosine, isoguanine, and basepairanalogs such as nitropyrrole and nitroindole, etc.

As described above generally for proteins, nucleic acid bioactive agentsmay be naturally occurring nucleic acids, random nucleic acids, or“biased” random nucleic acids. For example, digests of procaryotic oreukaryotic genomes may be used as is outlined above for proteins.

In general, probes of the present invention are designed to becomplementary to a target sequence (either the target analyte sequenceof the sample or to other probe sequences, as is described herein), suchthat hybridization of the target and the probes of the present inventionoccurs. This complementarity need not be perfect; there may be anynumber of base pair mismatches that will interfere with hybridizationbetween the target sequence and the single stranded nucleic acids of thepresent invention. However, if the number of mutations is so great thatno hybridization can occur under even the least stringent ofhybridization conditions, the sequence is not a complementary targetsequence. Thus, by “substantially complementary” herein is meant thatthe probes are sufficiently complementary to the target sequences tohybridize under the selected reaction conditions. High stringencyconditions are known in the art; see for example Maniatis et al.,Molecular Cloning: A Laboratory Manual, 2d Edition, 1989, and ShortProtocols in Molecular Biology, ed. Ausubel, et al., both of which arehereby incorporated by reference. Stringent conditions aresequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures. Anextensive guide to the hybridization of nucleic acids is found inTijssen, Techniques in Biochemistry and Molecular Biology—Hybridizationwith Nucleic Acid Probes, “Overview of principles of hybridization andthe strategy of nucleic acid assays” (1993). Generally, stringentconditions are selected to be about 5-10° C. lower than the thermalmelting point (T_(m)) for the specific sequence at a defined ionicstrength pH. The T_(m) is the temperature (under defined ionic strength,pH and nucleic acid concentration) at which 50% of the probescomplementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditionswill be those in which the salt concentration is less than about 1.0 Msodium ion, typically about 0.01 to 1.0 M sodium ion concentration (orother salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C. for short probes (e.g. 10 to 50 nucleotides) and at least about 60°C. for long probes (e.g. greater than 50 nucleotides). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide. In another embodiment, less stringenthybridization conditions are used; for example, moderate or lowstringency conditions may be used, as are known in the art; see Maniatisand Ausubel, supra, and Tijssen, supra.

The term “target sequence” or grammatical equivalents herein means anucleic acid sequence on a single strand of nucleic acid. The targetsequence may be a portion of a gene, a regulatory sequence, genomic DNA,cDNA, RNA including mRNA and rRNA, or others. It may be any length, withthe understanding that longer sequences are more specific. As will beappreciated by those in the art, the complementary target sequence maytake many forms. For example, it may be contained within a largernucleic acid sequence, i.e. all or part of a gene or mRNA, a restrictionfragment of a plasmid or genomic DNA, among others. As is outlined morefully below, probes are made to hybridize to target sequences todetermine the presence or absence of the target sequence in a sample.Generally speaking, this term will be understood by those skilled in theart.

In a preferred embodiment, the bioactive agents are organic chemicalmoieties, a wide variety of which are available in the literature.

In a preferred embodiment, each bead comprises a single type ofbioactive agent, although a plurality of individual bioactive agents arepreferably attached to each bead. Similarly, preferred embodimentsutilize more than one microsphere containing a unique bioactive agent;that is, there is redundancy built into the system by the use ofsubpopulations of microspheres, each microsphere in the subpopulationcontaining the same bioactive agent.

As will be appreciated by those in the art, the bioactive agents mayeither be synthesized directly on the beads, or they may be made andthen attached after synthesis. In a preferred embodiment, linkers areused to attach the bioactive agents to the beads, to allow both goodattachment, sufficient flexibility to allow good interaction with thetarget molecule, and to avoid undesirable binding reactions.

In a preferred embodiment, the bioactive agents are synthesized directlyon the beads. As is known in the art, many classes of chemical compoundsare currently synthesized on solid supports, such as peptides, organicmoieties, and nucleic acids. It is a relatively straightforward matterto adjust the current synthetic techniques to use beads.

In a preferred embodiment, the bioactive agents are synthesized first,and then covalently attached to the beads. As will be appreciated bythose in the art, this will be done depending on the composition of thebioactive agents and the beads. The functionalization of solid supportsurfaces such as certain polymers with chemically reactive groups suchas thiols, amines, carboxyls, etc. is generally known in the art.Accordingly, “blank” microspheres may be used that have surfacechemistries that facilitate the attachment of the desired functionalityby the user. Some examples of these surface chemistries for blankmicrospheres are listed in Table I.

TABLE I Surface chemistry Name: NH₂ Amine COOH Carboxylic Acid CHOAldehyde CH₂—NH₂ Aliphalic Amine CO NH₂ Amide CH₂—C1 ChloromethylCONH—NH₂ Hydrazide OH Hydroxyl SO₄ Sulfate SO₃ Sulfonate Ar NH₂ AromaticAmine

These functional groups can be used to add any number of differentbioactive agents to the beads, generally using known chemistries. Forexample, bioactive agents containing carbohydrates may be attached to anamino-functionalized support; the aldehyde of the carbohydrate is madeusing standard techniques, and then the aldehyde is reacted with anamino group on the surface. In an alternative embodiment, a sulfhydryllinker may be used. There are a number of sulfhydryl reactive linkersknown in the art such as SPDP, maleimides, α-haloacetyls, and pyridyldisulfides (see for example the 1994 Pierce Chemical Company catalog,technical section on cross-linkers, pages 155-200, incorporated hereinby reference) which can be used to attach cysteine containingproteinaceous agents to the support. Alternatively, an amino group onthe bioactive agent may be used for attachment to an amino group on thesurface. For example, a large number of stable bifunctional groups arewell known in the art, including homobifunctional and heterobifunctionallinkers (see Pierce Catalog and Handbook, pages 155-200). In anadditional embodiment, carboxyl groups (either from the surface or fromthe bioactive agent) may be derivatized using well known linkers (seethe Pierce catalog). For example, carbodiimides activate carboxyl groupsfor attack by good nucleophiles such as amines (see Torchilin et al.,Critical Rev. Therapeutic Drug Carrier Systems, 7(4):275-308 (1991),expressly incorporated herein). Proteinaceous bioactive agents may alsobe attached using other techniques known in the art, for example for theattachment of antibodies to polymers; see Slinkin et al., Bioconj. Chem.2:342-348 (1991); Torchilin et al., supra; Trubetskoy et al., Bioconj.Chem. 3:323-327 (1992); King et al., Cancer Res. 54:6176-6185 (1994);and Wilbur et al., Bioconjugate Chem. 5:220-235 (1994), all of which arehereby expressly incorporated by reference). It should be understoodthat the bioactive agents may be attached in a variety of ways,including those listed above. What is important is that manner ofattachment does not significantly alter the functionality of thebioactive agent; that is, the bioactive agent should be attached in sucha flexible manner as to allow its interaction with a target.

Specific techniques for immobilizing enzymes on microspheres are knownin the prior art. In one case, NH₂ surface chemistry microspheres areused. Surface activation is achieved with a 2.5% glutaraldehyde inphosphate buffered saline (10 mM) providing a pH of 6.9. (138 mM NaCl,2.7 mM, KCl). This is stirred on a stir bed for approximately 2 hours atroom temperature. The microspheres are then rinsed with ultrapure waterplus 0.01% tween 20 (surfactant) −0.02%, and rinsed again with a pH 7.7PBS plus 0.01% tween 20. Finally, the enzyme is added to the solution,preferably after being prefiltered using a 0.45 μm amicon micropurefilter.

In addition to a bioactive agent, the microspheres comprise an opticalsignature that can be used to identify the attached bioactive agent.That is, each subpopulation of microspheres comprise a unique opticalsignature or optical tag that can be used to identify the uniquebioactive agent of that subpopulation of microspheres; a bead comprisingthe unique optical signature may be distinguished from beads at otherlocations with different optical signatures. As is outlined herein, eachbioactive agent will have an associated unique optical signature suchthat any microspheres comprising that bioactive agent will beidentifiable on the basis of the signature. As is more fully outlinedbelow, it is possible to reuse or duplicate optical signatures within anarray, for example, when another level of identification is used, forexample when beads of different sizes are used, or when the array isloaded sequentially with different batches of beads.

In a preferred embodiment, the optical signature is generally a mixtureof reporter dyes, preferably fluorescent. By varying both thecomposition of the mixture (i.e. the ratio of one dye to another) andthe concentration of the dye (leading to differences in signalintensity), matrices of unique tags may be generated. This may be doneby covalently attaching the dyes to the surface of the beads, oralternatively, by entrapping the dye within the bead. The dyes may bechromophores or phosphors but are preferably fluorescent dyes, which dueto their strong signals provide a good signal-to-noise ratio fordecoding. Suitable dyes for use in the invention include, but are notlimited to, fluorescent lanthanide complexes, including those ofEuropium and Terbium, fluorescein, rhodamine, tetramethylrhodamine,eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green,stilbene, Lucifer Yellow, Cascade Blue™, Texas Red, and others describedin the 1989-1991 Molecular Probes Handbook by Richard P. Haugland,hereby expressly incorporated by reference.

In a preferred embodiment, the encoding can be accomplished in a ratioof at least two dyes, although more encoding dimensions may be added inthe size of the beads, for example. In addition, the labels aredistinguishable from one another; thus two different labels may comprisedifferent molecules (i.e. two different fluors) or, alternatively, onelabel at two different concentrations or intensity.

In a preferred embodiment, the dyes are covalently attached to thesurface of the beads. This may be done as is generally outlined for theattachment of the bioactive agents, using functional groups on thesurface of the beads. As will be appreciated by those in the art, theseattachments are done to minimize the effect on the dye.

In a preferred embodiment, the dyes are non-covalently associated withthe beads, generally by entrapping the dyes in the bead matrix or poresof the beads. Referring to the embodiment of FIG. 1, reporter dyes 14are added to the microsphere 10 with the encoding occurring in the ratioof two or more dyes. The reporter dyes 14 may be chromophore-type.Fluorescent dyes, however, are preferred because the strength of thefluorescent signal provides a better signal-to-noise ratio whendecoding. Additionally, encoding in the ratios of the two or more dyes,rather than single dye concentrations, is preferred since it providesinsensitivity to the intensity of light used to interrogate the reporterdye's signature and detector sensitivity.

In one embodiment, the dyes are added to the bioactive agent, ratherthan the beads, although this is generally not preferred.

FIG. 2 is a process diagram illustrating the preparation of themicrospheres. In step 50, an aliquot of stock microspheres are vacuumfiltered to produce a dry cake. In one implementation, microspherecopolymers of methylstyrene (87%) and divinylbenzene (13%) are used thathave a 3.1 micrometer (μm) diameter. The dry cake is then broken apartand a dye solution added to it in step 52 to encode optical signaturesof the microspheres with information concerning the intended surfacechemical functionalities. Dyes may be covalently bonded to themicrospheres' surface, but this consumes surface binding sites desirablyreserved for the chemical functionalities. Preferably, the microspheresare placed in a dye solution comprising a ratio of two or morefluorescent reporter dyes dissolved in an organic solvent that willswell the microspheres, e.g., dimethylformamide (DMF). The length oftime the microspheres are soaked in the dye solution will determinetheir intensity and the broadness of the ratio range.

In an exemplary two dye system, Texas Red Cadaverine (TRC) is used,which is excited at λ_(ab)=580 mm and emits at λ_(em)=630 mm, incombination with indodicarbocyanine (DilC): 610/670 (λ_(ab)/λ_(em)).Generally, dyes are selected to be compatible with the chemistriesinvolved in the analysis and to be spectrally compatible. This avoidsdeconvolution problems associated with determining signal contributionsbased on the presence of both the analyte and the encoding dye ratioscontributing to an overlapping emission spectral region.

Examples of other dyes that can be used are Oxazin (662/705), IR-144(745/825), IR-140 (776/882), IR-125 (786/800) from Exciton, and Bodipy665/676 from Molecular Probes, and Naphthofluorescein (605/675) alsofrom Molecular Probes. Lathanide complexes may also be used. Fluorescentdyes emitting in other than the near infrared may also be used.Chromophore dyes are still another alternative that produce an opticallyinterrogatable signature, as are more exotic formulations using Ramanscattering-based dyes or polarizing dyes, for example. The ability of aparticular dye pair to encode for different chemical functionalitiesdepends on the resolution of the ratiometric measurement.Conservatively, any dye pair should provide the ability to discriminateat least twenty different ratios. The number of unique combinations oftwo dyes made with a particular dye set is shown in the following TableII.

TABLE II Number of Combinations dyes in set possible 3 3 4 6 5 10 6 15

Thus, using six dyes and twenty distinct ratios for each dye pair, 300separate chemical functionalities may be encoded in a given populationof microspheres. Combining more than two dyes provides additionaldiversity in the encoding combinations. Furthermore, the concentrationof the dyes will contribute to their intensity; thus intensity isanother way to increase the number of unique optical signatures.

In step 54, the microspheres are vacuum filtered to remove excess dye.The microspheres are then washed in water or other liquid that does notswell the microspheres, but in which the dyes are still soluble. Thisallows the residual dye to be rinsed off without rinsing the dye out ofthe microspheres.

In step 56, the bioactive agent is attached to the microsphere surfaceif not already present. It should be understood that surface chemistriesmay be present throughout the microsphere's volume, and not limited tothe physical circumferential surface.

Once the microspheres are made comprising at least one bioactive agentand an optical signature, the microspheres are added to discrete siteson the surface of the substrate. This can be done in a number of ways,but generally comprises adding the beads to the surface under conditionsthat will allow the association of the microspheres on or at thediscrete sites. The association of the beads on the surface may comprisea covalent bonding of the bead to the surface, for example when chemicalattachment sites are added to both the substrate and the bead; anelectrostatic or hydroaffinity, when charge, hydrophobicity orhydrophilicity is used as the basis of the binding; a physical yetnon-covalent attachment such as the use of an adhesive; or a spatialattachment, for example the localization of a bead within a well. Insome embodiments it may be preferable to effect a more permanentattachment after the initial localization, for example through the useof cross-linking agents, a film or membrane over the array.

FIG. 3 schematically illustrates a microsphere system, or array ofmicrospheres, 100 formed from microsphere populations that havedifferent bioactive agents. While a large number of microspheres andbioactive agents may be employed, in this example only three microspherepopulations are shown. The individual populations, or subpopulations, ofmicrospheres are represented as 10 a,10 b,10 c carrying respectivebioactive agents or probe sequences 60 a,60 b,60 c, as exemplaryfunctionalities. The subpopulations may be combined in either a randomor ordered fashion on a substrate, with a corresponding distribution oftheir respective bioactive agents.

Typically, with conventional methods, mixing microsphere populationshaving different bioactive agents results in the loss of informationregarding the selectivity for each of the corresponding targetsequences. In a solution of microspheres with each of the probesequences 60 a, 60 b, and 60 c, it is possible to determine that atleast one of the target sequences 62 a, 62 b, and 62 c is present when afluorescent marker dye 64 concentration is observed on the microspheres10. However, with conventional approaches, typically there is no way todetermine which bioactive agent or probe sequence 60 a, 60 b, and 60 cis generating the activity since the information concerning whichmicrosphere contained which probe sequence was lost when thesubpopulations were mixed.

However, with the microsphere system 100 and method of the presentinvention, each microsphere in each subpopulation is encoded with acommon optical signature. In the illustrated example, the subpopulationrepresented by microsphere 10 a has a two reporter dye ratio of 10:1;the subpopulation of microspheres 10 b has a ratio of 1:1 of the samereporter dyes, and subpopulation of microspheres 10 c has a ratio of1:10 of the reporter dyes.

Thus, the randomly mixed subpopulations of microspheres are useful as ananalytic chemistry system based on each of the carried bioactive agents60 a-60 c separately. The microsphere array or system 100 is exposed toan analyte of interest to which some of the bioactive agents mayinteract. Any interaction changes the optical response of thecorresponding microspheres by, for example, binding a fluorescent dye 64to the microspheres. By identifying the chemical functionalities of themicrosphere in which the optical signature has changed, using theencoded dye combinations, information regarding the chemical identityand concentration of an analyte may be gained based upon the interactionor noninteraction of each bioactive agent contained in the microspheresystem 100.

The microspheres exhibiting activity or changes in their opticalsignature may be identified by a conventional optical train and opticaldetection system. Decoding can also be performed either manually orautomatically with the aid of a computer. Depending on the particularencoding or reporter dyes used and their operative wavelengths, opticalfilters designed for a particular wavelengths may be employed foroptical interrogation of the microspheres of bioactive agents. In apreferred embodiment, the analytic chemistry microsphere system is usedin conjunction with an optical fiber bundle or fiber optic array as asubstrate.

FIG. 4 is a schematic block diagram showing a microsphere-based analyticchemistry system employing a fiber optic assembly 200 with an opticaldetection system. The fiber optic assembly 200 comprises a fiber opticbundle or array 202, that is constructed from clad fibers so that lightdoes not mix between fibers. A microsphere array or system, 100 isattached to the bundle's distal end 212, with the proximal end 214 beingreceived by a z-translation stage 216 and x-y micropositioner 218. Thesetwo components act in concert to properly position the proximal end 214of the bundle 202 for a microscope objective lens 220. Light collectedby the objective lens 220 is passed to a reflected light fluorescenceattachment with three pointer cube slider 222. The attachment 222 allowsinsertion of light from a 75 Watt Xe lamp 224 through the objective lens220 to be coupled into the fiber bundle 202. The light from the source224 is condensed by condensing lens 226, then filtered and/or shutteredby filter and shutter wheel 228, and subsequently passes through a NDfilter slide 230.

Light returning from the distal end 212 of the bundle 202 is passed bythe attachment 222 to a magnification changer 232 which enablesadjustment of the image size of the fiber's proximal or distal end.Light passing through the magnification changer 232 is then shutteredand filtered by a second wheel 234. The light is then imaged on a chargecoupled device (CCD) camera 236. A computer 238 executes imagingprocessing software to process the information from the CCD camera 236and also possibly control the first and second shutter and filter wheels228, 234. The instrumentation exclusive of the fiber sensor 200, i.e.,to the left of the proximal end of the bundle 202 is discussed morecompletely by Bronk, et al., Anal. Chem. 1995, Vol. 67, number 17, pp.2750-2752.

The microsphere array or system 100 may be attached to the distal end ofthe optical fiber bundle using a variety of compatible processes. It isimportant that the microspheres are located close to the end of thebundle. This ensures that the light returning in each optical fiberpredominantly comes from only a single microsphere. This feature isnecessary to enable the interrogation of the optical signature ofindividual microspheres to identify reactions involving themicrosphere's functionality and also to decode the dye ratios containedin those microspheres. The adhesion or affixing technique, however, mustnot chemically insulate the microspheres from the analyte.

FIGS. 5A and 5B are micrographs of the distal end 212 of the bundle 202illustrating the preferred technique for attaching the microspheres 10to the bundle 202. Wells 250 are formed at the center of each opticalfiber 252 of the bundle 202. As shown in FIG. 5B, the size of the wells250 are coordinated with the size of the microspheres 10 so that themicrospheres 10 can be placed within the wells 250. Thus, each opticalfiber 252 of the bundle 202 conveys light from the single microsphere 10contained in 5 its well. Consequently, by imaging the end of the bundle202 onto the CCD array 236, the optical signatures of the microspheres10 are individually interrogatable.

FIG. 6 illustrates how the microwells 250 are formed and microspheres 10placed in the wells. A 1 mm hexagonally-packed imaging fiber containsapproximately 20,600 individual optical fibers that have coresapproximately 3.7 μm across (Part No. ET26 from Galileo Fibers).Typically, the cores of each fiber are hexagonally shaped as a resultthe starting preform; that is, during drawing the fiber does not usuallychange shape. In some cases, the shape can be circular, however.

In step 270, both the proximal and distal ends 212,214 of the fiberbundle 202 are successively polished on 12 μm, 9 μm, 3 μm, 1 μm, and 0.3μm lapping films. Subsequently, the ends can be inspected for scratcheson an atomic force microscope. In step 272, a representative etching isperformed on the distal end 212 of the bundle 202. A solution of 0.2grams NH₄F (ammonium fluoride) with 600 μl distilled H₂O and 100μ1 of HF(hydrofluoric acid), 50% stock solution, may be used. The distal end 212is etched in this solution for a specified time, preferablyapproximately 30 to 600 seconds, with about 80 seconds being preferred.

Upon removal from this solution, the bundle end is immediately placed indeionized water to stop any further etching in step 274. The fiber isthen rinsed in running tap water. At this stage, sonication ispreferably performed for several minutes to remove any salt productsfrom the reaction. The fiber is then allowed to air dry.

The foregoing procedure produces wells by the anisotropic etching of thefiber cores 254 favorably with respect to the cladding 256 for eachfiber of the bundle 202. The wells have approximately the diameter ofthe cores 254, 3.7 μm. This diameter is selected to be slightly largerthan the diameters of the microspheres used, 3.1 μm, in the example. Thepreferential etching occurs because the pure silica of the cores 254etches faster in the presence of hydrofluoric acid than thegermanium-doped silica claddings 256.

The microspheres are then placed in the wells 250 in step 276 accordingto a number of different techniques. The placement of the microspheresmay be accomplished by dripping a solution containing the desiredrandomly mixed subpopulations of the microspheres over the distal end212, sonicating the bundle to settle the microspheres in the wells, andallowing the microsphere solvent to evaporate. Alternatively, thesubpopulations could be added serially to the bundle end. Microspheres10 may then be fixed into the wells 250 by using a dilute solution ofsulfonated Nafion that is dripped over the end. Upon solventevaporation, a thin film of Nafion was formed over the microsphereswhich holds them in place. This approach is compatible for fixingmicrospheres for pH indication that carry FITC functionality. Theresulting array of fixed microspheres retains its pH sensitivity due tothe permeability of the sulfonated Nafion to hydrogen ions. Thisapproach, however, can not be employed generically as Nafion isimpermeable to most water soluble species. A similar approach can beemployed with different polymers. For example, solutions of polyethyleneglycol, polyacrylamide, or polyhydroxymethyl methacrylate (polyHEMA) canbe used in place of Nafion, providing the requisite permeability toaqueous species.

An alternative fixation approach employs microsphere swelling to entrapeach microsphere 10 in its corresponding microwell 250. In thisapproach, the microspheres are first distributed into the microwells 250by sonicating the microspheres suspended in a non-swelling solvent inthe presence of the microwell array on the distal end 212. Afterplacement into the microwells, the microspheres are subsequently exposedto an aqueous buffer in which they swell, thereby physically entrappingthem, analogous to muffins rising in a muffin tin.

In general, the methods of making the arrays and of decoding the arraysis done to maximize the number of different candidate agents that can beuniquely encoded. The compositions of the invention may be made in avariety of ways. In general, the arrays are made by adding a solution orslurry comprising the beads to a surface containing the sites forattachment of the beads. This may be done in a variety of buffers,including aqueous and organic solvents, and mixtures. The solvent canevaporate, and excess beads removed.

In a preferred embodiment, when non-covalent methods are used toassociate the beads to the array, a novel method of loading the beadsonto the array is used. This method comprises exposing the array to asolution of particles (including microspheres and cells) and thenapplying energy, e.g. agitating or vibrating the mixture. This resultsin an array comprising more tightly associated particles, as theagitation is done with sufficient energy to cause weakly-associatedbeads to fall off (or out, in the case of wells). These sites are thenavailable to bind a different bead. In this way, beads that exhibit ahigh affinity for the sites are selected. Arrays made in this way havetwo main advantages as compared to a more static loading: first of all,a higher percentage of the sites can be filled easily, and secondly, thearrays thus loaded show a substantial decrease in bead loss duringassays. Thus, in a preferred embodiment, these methods are used togenerate arrays that have at least about 50% of the sites filled, withat least about 75% being preferred, and at least about 90% beingparticularly preferred. Similarly, arrays generated in this mannerpreferably lose less than about 20% of the beads during an assay, withless than about 10% being preferred and less than about 5% beingparticularly preferred.

In this embodiment, the substrate comprising the surface with thediscrete sites is immersed into a solution comprising the particles(beads, cells, etc.). The surface may comprise wells, as is describedherein, or other types of sites on a patterned surface such that thereis a differential affinity for the sites. This differential affinityresults in a competitive process, such that particles that willassociate more tightly are selected. Preferably, the entire surface tobe “loaded” with beads is in fluid contact with the solution. Thissolution is generally a slurry ranging from about 10, 000:1beads:solution (vol:vol) to 1:1. Generally, the solution can compriseany number of reagents, including aqueous buffers, organic solvents,salts, other reagent components, etc. In addition, the solutionpreferably comprises an excess of beads; that is, there are more beadsthan sites on the array. Preferred embodiments utilize two-fold tobillion-fold excess of beads.

The immersion can mimic the assay conditions; for example, if the arrayis to be “dipped” from above into a microtiter plate comprising samples,this configuration can be repeated for the loading, thus minimizing thebeads that are likely to fall out due to gravity.

Once the surface has been immersed, the substrate, the solution, or bothare subjected to a competitive process, whereby the particles with loweraffinity can be disassociated from the substrate and replaced byparticles exhibiting a higher affinity to the site. This competitiveprocess is done by the introduction of energy, in the form of heat,sonication, stirring or mixing, vibrating or agitating the solution orsubstrate, or both.

A preferred embodiment utilizes agitation or vibration. In general, theamount of manipulation of the substrate is minimized to prevent damageto the array; thus, preferred embodiments utilize the agitation of thesolution rather than the array, although either will work. As will beappreciated by those in the art, this agitation can take on any numberof forms, with a preferred embodiment utilizing microtiter platescomprising bead solutions being agitated using microtiter plate shakers.

The agitation proceeds for a period of time sufficient to load the arrayto a desired fill. Depending on the size and concentration of the beadsand the size of the array, this time may range from about 1 second todays, with from about 1 minute to about 24 hours being preferred.

It should be noted that not all sites of an array may comprise a bead;that is, there may be some sites on the substrate surface which areempty. In addition, there may be some sites that contain more than onebead, although this is not preferred.

One of the most common microsphere formations is tentagel, astyrene-polyethylene glycol co-polymer. These microspheres are unswollenin nonpolar solvents such as hexane and swell approximately 20-40% involume upon exposure to a more polar or aqueous media. This approach isextremely desirable since it does not significantly compromise thediffusional or permeability properties of the microspheres themselves.

FIGS. 7A and 7B show polymer coated microspheres 12 in wells 250 aftertheir initial placement and then after tapping and exposure to airpulses. FIGS. 7A and 7B illustrate that there is no appreciable loss ofmicrospheres from the wells due to mechanical agitation even without aspecific fixing technique. This effect is probably due to electrostaticforces between the microspheres and the optical fibers. These forcestend to bind the microspheres within the wells. Thus, in mostenvironments, it may be unnecessary to use any chemical or mechanicalfixation for the microspheres.

In a preferred embodiment, particularly when wells are used, asonication step may be used to place beads in the wells.

It should be noted that not all sites of an array may comprise a bead;that is, there may be some sites on the substrate surface which areempty. In addition, there may be some sites that contain more than onebead, although this is not preferred.

In some embodiments, for example when chemical attachment is done, it ispossible to attach the beads in a non-random or ordered way. Forexample, using photoactivatible attachment linkers or photoactivatibleadhesives or masks, selected sites on the array may be sequentiallyrendered suitable for attachment, such that defined populations of beadsare laid down.

In addition, since the size of the array will be set by the number ofunique optical signatures, it is possible to “reuse” a set of uniqueoptical signatures to allow for a greater number of test sites. This maybe done in several ways; for example, by using a positional codingscheme within an array; different sub-bundles may reuse the set ofoptical signatures. Similarly, one embodiment utilizes bead size as acoding modality, thus allowing the reuse of the set of unique opticalsignatures for each bead size. Alternatively, sequential partial loadingof arrays with beads can also allow the reuse of optical signatures.

In a preferred embodiment, a spatial or positional coding system isdone. In this embodiment, there are sub-bundles or subarrays (i.e.portions of the total array) that are utilized. By analogy with thetelephone system, each subarray is an “area code”, that can have thesame tags (i.e. telephone numbers) of other subarrays, that areseparated by virtue of the location of the subarray. Thus, for example,the same unique tags can be reused from bundle to bundle. Thus, the useof 50 unique tags in combination with 100 different subarrays can forman array of 5000 different bioactive agents. In this embodiment, itbecomes important to be able to identify one bundle from another; ingeneral, this is done either manually or through the use of markerbeads, i.e. beads containing unique tags for each subarray.

In alternative embodiments, additional encoding parameters can be added,such as microsphere size. For example, the use of different size beadsmay also allow the reuse of sets of optical signatures; that is, it ispossible to use microspheres of different sizes to expand the encodingdimensions of the microspheres. Optical fiber arrays can be fabricatedcontaining pixels with different fiber diameters or cross-sections;alternatively, two or more fiber optic bundles, each with differentcross-sections of the individual fibers, can be added together to form alarger bundle; or, fiber optic bundles with fiber of the same sizecross-sections can be used, but just with different sized beads. Withdifferent diameters, the largest wells can be filled with the largestmicrospheres and then moving onto progressively smaller microspheres inthe smaller wells until all size wells are then filled. In this manner,the same dye ratio could be used to encode microspheres of differentsizes thereby expanding the number of different oligonucleotidesequences or chemical functionalities present in the array. Althoughoutlined for fiber optic substrates, this as well as the other methodsoutlined herein can be used with other substrates and with otherattachment modalities as well.

In a preferred embodiment, the coding and decoding is accomplished bysequential loading of the microspheres into the array. As outlined abovefor spatial coding, in this embodiment, the optical signatures can be“reused”. In this embodiment, the library of microspheres eachcomprising a different bioactive agent (or the subpopulations eachcomprise a different bioactive agent), is divided into a plurality ofsublibraries; for example, depending on the size of the desired arrayand the number of unique tags, 10 sublibraries each comprising roughly10% of the total library may be made, with each sublibrary comprisingroughly the same unique tags. Then, the first sublibrary is added to thefiber optic bundle comprising the wells, and the location of eachbioactive agent is determined, using its optical signature. The secondsublibrary is then added, and the location of each optical signature isagain determined. The signal in this case will comprise the “first”optical signature and the “second” optical signature; by comparing thetwo matrices the location of each bead in each sublibrary can bedetermined. Similarly, adding the third, fourth, etc. sublibrariessequentially will allow the array to be filled.

Thus, arrays are made of a large spectrum of chemical functionalitiesutilizing the compositions of invention comprising microspheres andsubstrates with discrete sites on a surface. Specifically, prior artsensors which can be adapted for use in the present invention includefour broad classifications of microsphere sensors: 1) basic indicatorchemistry sensors; 2) enzyme-based sensors; 3) immuno-based sensors(both of which are part of a broader general class of protein sensors);and 4) geno-sensors.

In a preferred embodiment, the bioactive agents are used to detectchemical compounds. A large number of basic indicator sensors have beenpreviously demonstrated. Examples include:

TABLE III TARGET ANALYTE Bioactive agent NOTES (λ_(AB)/λ_(EM)) pHSensors based on: seminaphthofluoresceins e.g., carboxyl-SNAFLseminaphthorhodafluors e.g., carboxyl-SNARF8-hydroxypyrene-1,3,6-trisulfonic acid Fluorescein CO2 Sensors based On:seminaphthofluoresceins e.g., carboxyl-SNAFL seminaphthorhodafluorse.g., carbody-SNARF 8-hydroxypyrene-1,3,6-trisulfonic acid Metal IonsSensors based on: desferriozamine B e.g., Fe cyclen derivative e.g., Cu,Zn derivatized peptides e.g., FITC-Gly-Gly-His, and FITC-Gly His, Cu, Znfluorexon (calcine) e.g., Ca, Mg, Cu, Pb, Ba calcine blue e.g., Ca, Mg,Cu methyl calcine blue e.g., Ca, Mg, Cu ortho-dianisidine tetraceticacid e.g., Zn (ODTA) bis-salicylidene ethylenediamine e.g., Al (SED)N-(6-methozy-8-quinolyl-p- e.g., Zn toluenesulfonamine (TSQ) Indo-1e.g., Mn, Ni Fura-2 e.g., Mn, Ni Magesium Green e.g., Mg, Cd, Tb O₂Siphenylisobenzofuran 409/476 Methoxyvinyl pyrene 352/401 Nitritediaminonaphthalene 340/377 NO Luminal 355/411 dihydrohodamine 289/noneCa²⁺ Bis-fura 340/380 Calcium Green visible light/530 Fura-2 340/380Indo-1 405/485 Fluo-3 visible light/525 Rhod-2 visible light/570 Mg²⁺Mag-Fura-2 340/380 Mag-Fura-5 340/380 Mag-Indo-1 405/485 Magnesium Green475/530 Magnesium Orange visible light/545 Zn²⁺ Newport Green 506/535TSQ Methoxy-Quinobyl 334/385 Cu⁺ Phen Green 492/517 Na⁺ SBFI 339/565SBFO 354/575 Sodium Green 506/535 K⁺ PBFI 336/557 Cl⁻ SPQ 344/443 MQAE350/460

Each of the chemicals listed in Table III directly produces an opticallyinterrogatable signal or a change in the optical signature, as is morefully outlined below, in the presence of the targeted analyte.

Enzyme-based microsphere sensors have also been demonstrated and couldbe manifest on microspheres. Examples include:

TABLE IV SENSOR TARGET Bioactive agent Glucose Sensor glucose oxidase(enz.) + O₂-sensitive dye (see Table I) Penicillin Sensor penicillinase(enz.) + pH-sensitive dye (see Table I) Urea Sensor urease (enz.) +pH-sensitive dye (see Table I) Acetylcholine Sensor acetylcholinesterase(enz.) + pH-sensitive dye (see Table I)

Generally, as more fully outlined above, the induced change in theoptical signal due to the presence of the enzyme-sensitive chemicalanalyte occurs indirectly in this class of chemical functionalities. Themicrosphere-bound enzyme, e.g., glucose oxidase, decomposes the targetanalyte, e.g., glucose, consume a co-substrate, e.g., oxygen, or producesome by-product, e.g., hydrogen peroxide. An oxygen sensitive dye isthen used to trigger the signal change. Thus, the product of thereaction is detected in a zone around the microsphere. That is, theproduct is not immobilized to the microsphere as is the enzyme, butrather the product is released from the microsphere-immobilized enzymeand detected in a region surrounding the microsphere.

Immuno-based microsphere sensors have been demonstrated for thedetection for environmental pollutants such as pesticides, herbicides,PCB's and PAH's. Additionally, these sensors have also been used fordiagnostics, such as bacterial (e.g., leprosy, cholera, lyme disease,and tuberculosis), viral (e.g., HIV, herpes simplex, cytomegalovirus),fungal (e.g., aspergillosis, candidiasis, cryptococcoses), Mycoplasmal(e.g., mycoplasmal pneumonia), Protozoal (e.g., amoebiasis,toxoplasmosis), Rickettsial (e.g., Rocky Mountain spotted fever), andpregnancy tests.

Microsphere genosensors may also be made (see the Examples). These aretypically constructed by attaching a probe sequence to the microspheresurface chemistry, typically via an NH₂ group. A fluorescent dyemolecule, e.g., fluorescein, is attached to the target sequence, whichis in solution. The optically interrogatable signal change occurs withthe binding of the target sequences to the microsphere. This produces ahigher concentration of dye surrounding the microsphere than in thesolution generally. A few demonstrated probe and target sequences, seeFerguson, J. A. et al. Nature Biotechnology, Vol. 14, December 1996, arelisted below in Table V.

TABLE V PROBE SEQUENCES TARGET SEQUENCES B-glo(+) (segment of humanB-globin)5′-NH₂- B-glo(+)-CF (CH₂)₈-)TT TTT TTT TCA ACT TCA TCC ACG5′-Fluorescein-TC AAC GTG GAT GAA GTT C- TTC ACC-3 3′ IFNG(interferongamma 1)5′-NH₂-(CH₂)₈-T₁₂- IFNG-CF TGG CTT CTC TTG GCT GTT ACT-3′5′-Fluorescein-AG TAA CAG CCA AGA GAA CCC AAA-3′IL2(interleukin-2)5′-NH₂-(CH₂)₈-T₁₂-TA ACC IL2-CF GAA TCC CAA ACT CACCAG-3′ 5′-Fluorescein-CT GGT GAG TTT GGG ATT CTT GTA-3′IL4(interleukin-4)5′NH₂-(CH₂)₈-T₁₂-CC AAC IL4-CF TGC TTC CCC CTC TGT-3′5′-Fluorescein-AC AGA GGG GGA AGC AGT TGG-3′IL6(interleukin-6)5′NH₂-(CH₂)₈-T₁₂-GT TGG IL6-CF GTC AGG GGT GGT TATT-3′ 5′-Fluorescein-AA TAA CCA CCC CTG ACC CAA C-3′

It should be further noted that the genosensors can be based on the useof hybridization indicators as the labels. Hybridization indicatorspreferentially associate with double stranded nucleic acid, usuallyreversibly. Hybridization indicators include intercalators and minorand/or major groove binding moieties. In a preferred embodiment,intercalators may be used; since intercalation generally only occurs inthe presence of double stranded nucleic acid, only in the presence oftarget hybridization will the label light up.

The present invention may be used with any or all of these types ofsensors. As will be appreciated by those in the art, the type andcomposition of the sensor will vary widely, depending on the compositionof the target analyte. That is, sensors may be made to detect nucleicacids, proteins (including enzyme sensors and immunosensors), lipids,carbohydrates, etc; similarly, these sensors may include bioactiveagents that are nucleic acids, proteins, lipids, carbohydrates, etc. Inaddition, a single array sensor may contain different binding ligandsfor multiple types of analytes; for example, an array sensor for HIV maycontain multiple nucleic acid probes for direct detection of the viralgenome, protein binding ligands for direct detection of the viralparticle, immuno-components for the detection of anti-HIV antibodies,etc.

In addition to the beads and the substrate, the compositions of theinvention may include other components, such as light sources, opticalcomponents such as lenses and filters, detectors, computer componentsfor data analysis, etc.

The arrays of the present invention are constructed such thatinformation about the identity of the bioactive agent is built into thearray, such that the random deposition of the beads on the surface ofthe substrate can be “decoded” to allow identification of the bioactiveagent at all positions. This may be done in a variety of ways.

In a preferred embodiment, the beads are loaded onto the substrate andthen the array is decoded, prior to running the assay. This is done bydetecting the optical signature associated with the bead at each site onthe array. This may be done all at once, if unique optical signaturesare used, or sequentially, as is generally outlined above for the“reuse” of sets of optical signatures. Alternatively, decoding may occurafter the assay is run.

Once made and decoded if necessary, the compositions find use in anumber of applications. Generally, a sample containing a target analyte(whether for detection of the target analyte or screening for bindingpartners of the target analyte) is added to the array, under conditionssuitable for binding of the target analyte to at least one of thebioactive agents, i.e. generally physiological conditions. The presenceor absence of the target analyte is then detected. As will beappreciated by those in the art, this may be done in a variety of ways,generally through the use of a change in an optical signal. This changecan occur via many different mechanisms. A few examples include thebinding of a dye-tagged analyte to the bead, the production of a dyespecies on or near the beads, the destruction of an existing dyespecies, a change in the optical signature upon analyte interaction withdye on bead, or any other optical interrogatable event.

In a preferred embodiment, the change in optical signal occurs as aresult of the binding of a target analyte that is labeled, eitherdirectly or indirectly, with a detectable label, preferably an opticallabel such as a fluorochrome. Thus, for example, when a proteinaceoustarget analyte is used, it may be either directly labeled with a fluor,or indirectly, for example through the use of a labeled antibody.Similarly, nucleic acids are easily labeled with fluorochromes, forexample during PCR amplification as is known in the art. Alternatively,upon binding of the target sequences, an intercalating dye (e.g.,ethidium bromide) can be added subsequently to signal the presence ofthe bound target to the probe sequence. Upon binding of the targetanalyte to a bioactive agent, there is a new optical signal generated atthat site, which then may be detected.

Alternatively, in some cases, as discussed above, the target analytesuch as an enzyme generates a species (for example, a fluorescentproduct) that is either directly or indirectly detectable optically.

Furthermore, in some embodiments, a change in the optical signature maybe the basis of the optical signal. For example, the interaction of somechemical target analytes with some fluorescent dyes on the beads mayalter the optical signature, thus generating a different optical signal.For example, fluorophore derivatized receptors may be used in which thebinding of the ligand alters the signal.

In a preferred embodiment, sensor redundancy is used. In thisembodiment, a plurality of sensor elements, e.g. beads, comprisingidentical bioactive agents are used. That is, each subpopulationcomprises a plurality of beads comprising identical bioactive agents(e.g. binding ligands). By using a number of identical sensor elementsfor a given array, the optical signal from each sensor element can becombined and any number of statistical analyses run, as outlined below.This can be done for a variety of reasons. For example, in time varyingmeasurements, redundancy can significantly reduce the noise in thesystem. For non-time based measurements, redundancy can significantlyincrease the confidence of the data.

In a preferred embodiment, a plurality of identical sensor elements areused. As will be appreciated by those in the art, the number ofidentical sensor elements will vary with the application and use of thesensor array. In general, anywhere from 2 to thousands may be used, withfrom 2 to 100 being preferred, 2 to 50 being particularly preferred andfrom 5 to 20 being especially preferred. In general, preliminary resultsindicate that roughly 10 beads gives a sufficient advantage, althoughfor some applications, more identical sensor elements can be used.

Once obtained, the optical response signals from a plurality of sensorbeads within each bead subpopulation can be manipulated and analyzed ina wide variety of ways, including baseline adjustment, averaging,standard deviation analysis, distribution and cluster analysis,confidence interval analysis, mean testing, etc.

In a preferred embodiment, the first manipulation of the opticalresponse signals is an optional baseline adjustment. In a typicalprocedure, the standardized optical responses are adjusted to start at avalue of 0.0 by subtracting the integer 1.0 from all data points. Doingthis allows the baseline-loop data to remain at zero even when summedtogether and the random response signal noise is canceled out. When thesample is a vapor, the vapor pulse-loop temporal region, however,frequently exhibits a characteristic change in response, eitherpositive, negative or neutral, prior to the vapor pulse and oftenrequires a baseline adjustment to overcome noise associated with driftin the first few data points due to charge buildup in the CCD camera. Ifno drift is present, typically the baseline from the first data pointfor each bead sensor is subtracted from all the response data for thesame bead. If drift is observed, the average baseline from the first tendata points for each bead sensor is subtracted from the all the responsedata for the same bead. By applying this baseline adjustment, whenmultiple bead responses are added together they can be amplified whilethe baseline remains at zero. Since all beads respond at the same timeto the sample (e.g. the vapor pulse), they all see the pulse at theexact same time and there is no registering or adjusting needed foroverlaying their responses. In addition, other types of baselineadjustment may be done, depending on the requirements and output of thesystem used.

Once the baseline has been adjusted, although in some embodiments thisis not required, a number of possible statistical analyses may be run togenerate known statistical parameters. Analyses based on redundancy areknown and generally described in texts such as Freund and Walpole,Mathematical Statistics, Prentice Hall, Inc. New Jersey, 1980, herebyincorporated by reference in its entirety.

In a preferred embodiment, signal summing is done by simply adding theintensity values of all responses at each time point, generating a newtemporal response comprised of the sum of all bead responses. Thesevalues can be baseline-adjusted or raw. As for all the analysesdescribed herein, signal summing can be performed in real time or duringpost-data acquisition data reduction and analysis. In one embodiment,signal summing is performed with a commercial spreadsheet program(Excel, Microsoft, Redmond, Wash.) after optical response data iscollected.

In a preferred embodiment, cumulative response data is generated bysimply adding all data points in successive time intervals. This finalcolumn, comprised of the sum of all data points at a particular timeinterval, may then be compared or plotted with the individual beadresponses to determine the extent of signal enhancement or improvedsignal-to-noise ratios as shown in FIGS. 14 and 15.

In a preferred embodiment, the mean of the subpopulation (i.e. theplurality of identical beads) is determined, using the well knownEquation 1:

$\begin{matrix}{\mu = {\sum\frac{x_{i}}{n}}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

In some embodiments, the subpopulation may be redefined to exclude somebeads if necessary (for example for obvious outliers, as discussedbelow).

In a preferred embodiment, the standard deviation of the subpopulationcan be determined, generally using Equation 2 (for the entiresubpopulation) and Equation 3 (for less than the entire subpopulation):

$\begin{matrix}{\sigma = \sqrt{\frac{\sum\left( {x_{i} - \mu} \right)^{2}}{n}}} & {{Equation}\mspace{14mu} 2} \\{s = \sqrt{\frac{\sum\left( {x_{i} - \overset{\_}{x}} \right)^{2}}{n - 1}}} & {{Equation}\mspace{14mu} 3}\end{matrix}$

As for the mean, the subpopulation may be redefined to exclude somebeads if necessary (for example for obvious outliers, as discussedbelow).

In a preferred embodiment, statistical analyses are done to evaluatewhether a particular data point has statistical validity within asubpopulation by using techniques including, but not limited to, tdistribution and cluster analysis. This may be done to statisticallydiscard outliers that may otherwise skew the result and increase thesignal-to-noise ratio of any particular experiment. This may be doneusing Equation 4:

$\begin{matrix}{t = \frac{\overset{\_}{x} - \mu}{s/\sqrt{n}}} & {{Equation}\mspace{14mu} 4}\end{matrix}$

In a preferred embodiment, the quality of the data is evaluated usingconfidence intervals, as is known in the art. Confidence intervals canbe used to facilitate more comprehensive data processing to measure thestatistical validity of a result.

In a preferred embodiment, statistical parameters of a subpopulation ofbeads are used to do hypothesis testing. One application is testsconcerning means, also called mean testing. In this application,statistical evaluation is done to determine whether two subpopulationsare different. For example, one sample could be compared with anothersample for each subpopulation within an array to determine if thevariation is statistically significant.

In addition, mean testing can also be used to differentiate twodifferent assays that share the same code. If the two assays giveresults that are statistically distinct from each other, then thesubpopulations that share a common code can be distinguished from eachother on the basis of the assay and the mean test, shown below inEquation 5:

$\begin{matrix}{z = \frac{{\overset{\_}{x}}_{1} - {\overset{\_}{x}}_{2}}{\sqrt{\frac{\sigma_{1}^{2}}{n_{1}} + \frac{\sigma_{2}^{2}}{n_{2}}}}} & {{Equation}\mspace{14mu} 5}\end{matrix}$

Furthermore, analyzing the distribution of individual members of asubpopulation of sensor elements may be done. For example, asubpopulation distribution can be evaluated to determine whether thedistribution is binomial, Poisson, hypergeometric, etc.

In addition to the sensor redundancy, a preferred embodiment utilizes aplurality of sensor elements that are directed to a single targetanalyte but yet are not identical. For example, a single target nucleicacid analyte may have two or more sensor elements each comprising adifferent probe. This adds a level of confidence as non-specific bindinginteractions can be statistically minimized. When nucleic acid targetanalytes are to be evaluated, the redundant nucleic acid probes may beoverlapping, adjacent, or spatially separated. However, it is preferredthat two probes do not compete for a single binding site, so adjacent orseparated probes are preferred. Similarly, when proteinaceous targetanalytes are to be evaluated, preferred embodiments utilize bioactiveagent binding agents that bind to different parts of the target. Forexample, when antibodies (or antibody fragments) are used as bioactiveagents for the binding of target proteins, preferred embodiments utilizeantibodies to different epitopes.

In this embodiment, a plurality of different sensor elements may beused, with from about 2 to about 20 being preferred, and from about 2 toabout 10 being especially preferred, and from 2 to about 5 beingparticularly preferred, including 2, 3, 4 or 5. However, as above, moremay also be used, depending on the application.

As above, any number of statistical analyses may be run on the data fromtarget redundant sensors.

One benefit of the sensor element summing (referred to herein as “beadsumming” when beads are used), is the increase in sensitivity that canoccur. Detection limits in the zeptomole range can be observed.

As will be appreciated by those in the art, in some embodiments, thepresence or absence of the target analyte may be determined usingchanges in other optical or non-optical signals, including, but notlimited to, surface enhanced Raman spectroscopy, surface plasmonresonance, radioactivity, etc.

The assays may be run under a variety of experimental conditions, aswill be appreciated by those in the art. A variety of other reagents maybe included in the screening assays. These include reagents like salts,neutral proteins, e.g. albumin, detergents, etc which may be used tofacilitate optimal protein-protein binding and/or reduce non-specific orbackground interactions. Also reagents that otherwise improve theefficiency of the assay, such as protease inhibitors, nucleaseinhibitors, anti-microbial agents, etc., may be used. The mixture ofcomponents may be added in any order that provides for the requisitebinding. Various blocking and washing steps may be utilized as is knownin the art.

In a preferred embodiment, the compositions are used to probe a samplesolution for the presence or absence of a target analyte. By “targetanalyte” or “analyte” or grammatical equivalents herein is meant anyatom, molecule, ion, molecular ion, compound or particle to be eitherdetected or evaluated for binding partners. As will be appreciated bythose in the art, a large number of analytes may be used in the presentinvention; basically, any target analyte can be used which binds abioactive agent or for which a binding partner (i.e. drug candidate) issought.

Suitable analytes include organic and inorganic molecules, includingbiomolecules. When detection of a target analyte is done, suitabletarget analytes include, but are not limited to, an environmentalpollutant (including pesticides, insecticides, toxins, etc.); a chemical(including solvents, polymers, organic materials, etc.); therapeuticmolecules (including therapeutic and abused drugs, antibiotics, etc.);biomolecules (including hormones, cytokines, proteins, nucleic acids,lipids, carbohydrates, cellular membrane antigens and receptors (neural,hormonal, nutrient, and cell surface receptors) or their ligands, etc);whole cells (including procaryotic (such as pathogenic bacteria) andeukaryotic cells, including mammalian tumor cells); viruses (includingretroviruses, herpesviruses, adenoviruses, lentiviruses, etc.); andspores; etc. Particularly preferred analytes are nucleic acids andproteins.

In a preferred embodiment, the target analyte is a protein. As will beappreciated by those in the art, there are a large number of possibleproteinaceous target analytes that may be detected or evaluated forbinding partners using the present invention. Suitable protein targetanalytes include, but are not limited to, (1) immunoglobulins; (2)enzymes (and other proteins); (3) hormones and cytokines (many of whichserve as ligands for cellular receptors); and (4) other proteins.

In a preferred embodiment, the target analyte is a nucleic acid. Theseassays find use in a wide variety of applications.

In a preferred embodiment, the probes are used in genetic diagnosis. Forexample, probes can be made using the techniques disclosed herein todetect target sequences such as the gene for nonpolyposis colon cancer,the BRCA1 breast cancer gene, P53, which is a gene associated with avariety of cancers, the Apo E4 gene that indicates a greater risk ofAlzheimer's disease, allowing for easy presymptomatic screening ofpatients, mutations in the cystic fibrosis gene, or any of the otherswell known in the art.

In an additional embodiment, viral and bacterial detection is done usingthe complexes of the invention. In this embodiment, probes are designedto detect target sequences from a variety of bacteria and viruses. Forexample, current blood-screening techniques rely on the detection ofanti-HIV antibodies. The methods disclosed herein allow for directscreening of clinical samples to detect HIV nucleic acid sequences,particularly highly conserved HIV sequences. In addition, this allowsdirect monitoring of circulating virus within a patient as an improvedmethod of assessing the efficacy of anti-viral therapies. Similarly,viruses associated with leukemia, HTLV-I and HTLV-II, may be detected inthis way. Bacterial infections such as tuberculosis, clymidia and othersexually transmitted diseases, may also be detected.

In a preferred embodiment, the nucleic acids of the invention find useas probes for toxic bacteria in the screening of water and food samples.For example, samples may be treated to lyse the bacteria to release itsnucleic acid, and then probes designed to recognize bacterial strains,including, but not limited to, such pathogenic strains as, Salmonella,Campylobacter, Vibrio cholerae, Leishmania, enterotoxic strains of E.coli, and Legionnaire's disease bacteria. Similarly, bioremediationstrategies may be evaluated using the compositions of the invention.

In a further embodiment, the probes are used for forensic “DNAfingerprinting” to match crime-scene DNA against samples taken fromvictims and suspects.

In an additional embodiment, the probes in an array are used forsequencing by hybridization.

The present invention also finds use as a methodology for the detectionof mutations or mismatches in target nucleic acid sequences. Forexample, recent focus has been on the analysis of the relationshipbetween genetic variation and phenotype by making use of polymorphic DNAmarkers. Previous work utilized short tandem repeats (STRs) aspolymorphic positional markers; however, recent focus is on the use ofsingle nucleotide polymorphisms (SNPs), which occur at an averagefrequency of more than 1 per kilobase in human genomic DNA. Some SNPs,particularly those in and around coding sequences, are likely to be thedirect cause of therapeutically relevant phenotypic variants. There area number of well known polymorphisms that cause clinically importantphenotypes; for example, the apoE2/3/4 variants are associated withdifferent relative risk of Alzheimer's and other diseases (see Cordor etal., Science 261 (1993). Multiplex PCR amplification of SNP loci withsubsequent hybridization to oligonucleotide arrays has been shown to bean accurate and reliable method of simultaneously genotyping at leasthundreds of SNPs; see Wang et al., Science, 280:1077 (1998); see alsoSchafer et al., Nature Biotechnology 16:33-39 (1998). The compositionsof the present invention may easily be substituted for the arrays of theprior art.

In a preferred embodiment, the compositions of the invention are used toscreen bioactive agents to find an agent that will bind, and preferablymodify the function of, a target molecule. As above, a wide variety ofdifferent assay formats may be run, as will be appreciated by those inthe art. Generally, the target analyte for which a binding partner isdesired is labeled; binding of the target analyte by the bioactive agentresults in the recruitment of the label to the bead, with subsequentdetection.

In a preferred embodiment, the binding of the bioactive agent and thetarget analyte is specific; that is, the bioactive agent specificallybinds to the target analyte. By “specifically bind” herein is meant thatthe agent binds the analyte, with specificity sufficient todifferentiate between the analyte and other components or contaminantsof the test sample. However, as will be appreciated by those in the art,it will be possible to detect analytes using binding which is not highlyspecific; for example, the systems may use different binding ligands,for example an array of different ligands, and detection of anyparticular analyte is via its “signature” of binding to a panel ofbinding ligands, similar to the manner in which “electronic noses” work.This finds particular utility in the detection of chemical analytes. Thebinding should be sufficient to remain bound under the conditions of theassay, including wash steps to remove non-specific binding, although insome embodiments, wash steps are not desired; i.e. for detecting lowaffinity binding partners. In some embodiments, for example in thedetection of certain biomolecules, the dissociation constants of theanalyte to the binding ligand will be less than about 10⁻⁴-10⁻⁶ M⁻¹,with less than about 10⁻⁵ to 10⁻⁹ M⁻¹ being preferred and less thanabout 10⁻⁷-10⁻⁹ M⁻¹ being particularly preferred.

As described herein, in addition to the use of the sensors for detectinglabeled analytes, the invention also provides for the detection oftarget analytes that affect enzymatic reactions. In general, thisembodiment can be described as follows.

In one aspect the invention provides a method of detecting a targetanalyte in a sample. In one embodiment the target analyte is a moleculethat modulates the activity of an enzyme. Alternatively, the targetanalyte is itself an enzyme. In addition the invention provides areaction component that is immobilized at a discrete site on an array.

By “reaction component” is meant a molecule that affects a reaction,when contacted with other molecules. In a preferred embodiment thereaction is an enzymatic reaction, although it could also includechemical or binding reactions. By “affects” a reaction is meant toinclude but is not limited to inducing, activating, altering, forexample slowing down or speeding up a reaction, or inhibiting areaction.

By “immobilized” is meant to affix to a site on an array. In oneembodiment immobilization is direct. That is, the reaction component isdirectly attached to the array. In an alternative embodiment,immobilization is indirect. In this embodiment, the reaction componentis attached to the array through an intermediate moiety such as amicrosphere. The reaction component is attached to the microsphere orarray by any of the methods described herein.

When the target analyte is an enzyme, the reaction component modulatesthe activity of the enzyme. In a preferred embodiment the reactioncomponent is a substrate for the enzyme, i.e. “enzyme substrate”. Whenreferring to enzyme substrates or substrates of an enzyme, the skilledartisan will recognize that this refers to a molecule that is convertedby an enzyme into a product, as a result of an enzymatic reaction. Inthis embodiment, the array may include multiple discrete reactioncomponents at different sites on the array. In one embodiment only someof the reaction components are substrates for the enzyme. Than is, asubset or subsets of reaction components are substrates for the enzyme.

Alternatively, the enzyme is immobilized at the discrete site, and atarget analyte modulates the activity of the enzyme (the reactioncomponent in this configuration). As is known in the art, molecules thatmodulate the activity of enzymes include but are not limited tosubstrates, co-factors, ligands, agonists, antagonists, inhibitors, andthe like.

In one aspect, the invention provides a method for detecting the productof the enzymatic reaction as an indication of the presence of the enzymeand/or enzyme modulator. That is, upon enzyme activation, a product ofthe reaction is released from the array where it is detected. In apreferred embodiment, the product of the enzymatic reaction is detectedupon its release from the enzyme in a zone around the discrete sitewhere the enzyme and/or target analyte is located. That is, the productis detected in a zone surrounding the site on the array. By “around” or“surrounding” or grammatical equivalents herein is meant near thediscrete site on the array. That is, the product is detected remote fromor released from the discrete site. While it is appreciated that in someinstances not all of the product will be released from the array, in apreferred embodiment substantially all of the product, once convertedfrom the enzyme substrate, is released from the array. By “substantiallyall” is meant at least about 60 percent, preferably more than about 75percent and most preferably more than about 85 percent.

As such, the product is detected not while immobilized, but rather whilediffusing from the site. The product can be detected either directly orindirectly. That is, the released product either contains a detectablelabel or is otherwise directly detectable. Alternatively, the productcan be detected indirectly. In this embodiment, the product binds to orassociates with other molecules that produce or result in a detectablesignal. In a preferred embodiment, the product is a substrate for amolecule such as an enzyme. When the product of the reaction is asubstrate for a subsequent enzyme, the subsequent enzyme is a detectionmolecule as described herein.

Accordingly, in a preferred embodiment the invention provides a methodof detecting a target analyte in a sample. The method includes providingan array as described herein. The array contains discrete sites to whichreaction component(s) are attached. In some embodiments, the reactioncomponent(s) are covalently attached. The array is contacted with thesample containing a target analyte and the product of an enzymaticreaction is detected in a region or zone around the site, as anindication of the presence of the target analyte.

In one embodiment the target analyte is an enzyme. As such, the samplescontain at least one enzyme. In this embodiment, the reaction componentis a substrate of the enzyme, or in an alternative embodiment, thereaction component is a co-factor. When the reaction component is not asubstrate, the appropriate substrate is added to the array, for examplein solution phase. In one embodiment each site contains a reactioncomponent, although in another embodiment, each site includes at leasttwo reaction components. That is, multiple reaction components are addedin a single site.

In an alternative embodiment the target analyte is an enzyme substrate.As such, the reaction component is an enzyme. Alternatively, when thereaction component is an enzyme, the target analyte is an enzymeinhibitor.

The attachment of reaction components including enzymes or enzymesubstrates to array sites, particularly beads, is outlined herein andwill be appreciated by those in the art. In general, the use of flexiblelinkers is preferred, as this allows reaction components to interactwith target analytes. However, for some types of attachment, linkers arenot needed. Attachment proceeds on the basis of the composition of thearray site (i.e. either the substrate or the bead, depending on whicharray system is used) and the composition of the enzyme. In a preferredembodiment, depending on the composition of the array site (e.g. thebead), it will contain chemical functional groups for subsequentattachment of other moieties. For example, beads comprising a variety ofchemical functional groups such as amines are commercially available.Preferred functional groups for attachment are amino groups, carboxygroups, oxo groups and thiol groups, with amino groups beingparticularly preferred. Using these functional groups, the enzymes canbe attached using functional groups on the enzymes. For example, enzymescontaining amino groups can be attached to particles comprising aminogroups, for example using linkers as are known in the art; for example,homo- or hetero-bifunctional linkers as are well known (see 1994 PierceChemical Company catalog, technical section on cross-linkers, pages155-200, incorporated herein by reference). In one embodiment thereaction component is attached via a cleavable linker. By “cleavablelinker” is meant a linker designed and intended to be specificallycleaved. Examples of cleavable linkers include photocleavable linkers,acid-cleavable linkers, and the like. Examples of cleavable linkers areoutlined in more detail in U.S. Pat. No. 5,856,083, which is herebyexpressly incorporated by reference.

In an alternative embodiment, at least one of the reaction components isnon-cleavable. By “non-cleavable linker” is meant a linker that is notdesigned or intended to be specifically cleaved. That is, while thenon-cleavable linkers are not resistant to all cleavage agents, thelinker is not used so that the molecule to which it is attached can bereleased from the array. Thus, in contrast to the linkers as describedin U.S. Pat. No. 5,856,063, the use of non-cleavable linkers to attachreaction components to the array results in a stable attachment of thereaction molecules.

In one embodiment the array comprises microspheres to which the reactioncomponent is attached or reaction components are attached. Microspheresare distributed on the discrete sites on the array as described herein.However, other types of arrays are well known and can be used in thisformat; spotted, printed or photolithographic arrays are well known; seefor example WO 95/25116; WO 95/35505; PCT US98/09163; U.S. Pat. Nos.5,700,637; 5,807,522 and 5,445,934; and U.S. Ser. Nos. 08/851,20309/187,289; and references cited within, all of which are expresslyincorporated by reference. If beads are not used, preferred embodimentsutilize a spotted array.

In some instances only one of the reactants (i.e. reaction components)is immobilized on the discrete site. As such, any other necessaryreactants or co-factors are added to the immobilized reactant forexample, in solution phase. The product of the reaction is released fromthe site into the area surrounding the site where it is detected by anyof the detection methods as described herein.

In one embodiment, the target analyte is detected in a zone surroundinga discrete site on an array. That is, the target analyte is notimmobilized or attached to the microsphere, but rather is detected in aregion surrounding the discrete site.

In an alternative embodiment, the product of the reaction between thetarget analyte and the reaction component is detected in a zonesurrounding a discrete site on an array. That is, following theenzymatic reaction, the product(s) is released from the immobilizedreaction components. Thus, it is detected in a zone surrounding thesite.

In one embodiment, when detecting a product in a zone surrounding adiscrete site on an array, the reaction takes place in the presence ofsolutions and/or a matrix to slow down diffusion of the product from thesite. That is, the reaction takes place in the presence of a diffusionretardant. In this respect, the product maintains an increased localconcentration in the zone around the site for increased time. Solutionsto diminish the diffusion of the product should not interfere thereaction between the reactants and include but are not limited toglycerol, polyethylene glycol, agarose, agar, polyacrylamide and otherpolymers. In a preferred embodiment the polymer is readily varied in itsconcentration so that the rate of diffusion of the product is adjustedas is necessary.

As one of skill in the art recognizes, enzymes that can be screenedinclude but are not limited to polymerases, ligases, proteases includingcaspases, nucleotide cyclases, ribozymes, restriction endonucleases,transferases, lipases, and the like.

In one embodiment, the immobilized reactants correspond to knownsubstrates of enzymes. As such, samples are screened for the presence ofthe particular enzyme by detecting a change in the respectiveimmobilized substrate. In a preferred embodiment, a subpopulation ofsites comprises discrete substrates for a plurality of enzymes. That is,a subpopulation contains substrates for a particular enzyme, yet thearray may contain a population of microspheres that includessubpopulations that contain substrates for a number of enzymes. Thearray is contacted with a solution to be screened, for example, a tissueextract. A reaction between an enzyme and its respective substrate isdetected as the product of the reaction is detected in the zone aroundthe site containing the substrate molecule.

In an alternative embodiment a plurality of immobilized reactants aresubstrates for the same enzyme. That is, multiple sites on an arraycontain substrates for the same enzyme. In a preferred embodiment, thesubstrates are different. In one embodiment the substrates are differentwhile remaining substrates for the same enzyme. For example, an arraymay contain multiple different oligonucleotides at different sites on anarray. The oligonucleotides may contain unique or different sequences.However, all of the oligonucleotides are substrates for a polymerase ornuclease.

In one embodiment each microsphere contains multiple reactioncomponents. That is, as is known in the art, certain enzymes requiremultiple substrates and/or cofactors. Accordingly, in this embodiment, amicrosphere may contain a plurality of reaction components. The reactioncomponents may include an enzyme, substrate, co-factor or other moleculethat effects the reaction.

In one embodiment, the substrates are tagged or labeled such that thereleased product contains an identifier that is detectable, for examplea fluorescent label. In a preferred embodiment, the substrates contain afluorescent label that is quenched in the immobilized uncleavedsubstrate; however, upon cleavage, the released product containing thefluorescent label is unquenched and thus, emits a fluorescent signal inthe zone surrounding the bead.

In an alternative embodiment, the released product is not directlydetectable, but rather is indirectly detected by binding agents such asantibodies, or complementary nucleic acid probes. In this embodiment,the binding agents comprise the label.

In one embodiment the reaction product is a substrate for a detectionmolecule that may or may not be attached to the microspheres. By“detection molecule” is meant a molecule that reacts with the productfrom the first reaction to produce either directly or indirectly adetectable signal. When the detection molecule is not attached to thebeads, it is added to the microsphere either prior to, simultaneouslywith or following the addition of the first mobile-phase reactant. Whenthe detection molecule is immobilized to the microsphere it interactswith the reaction product following the first reaction. In thisembodiment a microsphere may contain a reaction component and adetection molecule. Accordingly, in a preferred embodiment the productof the reaction with the reaction component is detected by the detectionmolecule.

In one embodiment the detection molecule is an enzyme that catalyzes areaction, the product of which is directly detectable or as is wellknown in the art is converted to a substance that produces a detectablesignal. As one of skill in the art appreciates, examples of detectionmolecules include but are not limited to b-galactosidase, fireflyluciferase and the like.

In a preferred embodiment, particularly when secondary enzymes(detection molecules) are used in the reaction, the enzyme(s) may beattached, preferably through the use of flexible linkers, to the siteson the array, e.g. the beads, as described herein. For example, whenpyrosequencing is done, one embodiment utilizes detection based on thegeneration of a chemiluminescent signal in the “zone” around the bead.Pyrosequencing is described in more detail in U.S. Ser. Nos. 60/130,089,60/160,927, 09/513,362, 60/135,053, 09/425,633, 09/535, 854, 09/553,993and 09/556,463, all of which are hereby expressly incorporated byreference in their entirety.

Pyrosequencing is an extension method that can be used to add one ormore nucleotides to the 3′-terminus of an oligonucleotide.Pyrosequencing relies on the detection of a reaction product, PPi,produced during the addition of an NTP to a growing oligonucleotidechain, rather than on a label attached to the nucleotide. One moleculeof PPi is produced per dNTP added to the extension primer. Accordingly,by running sequential reactions with each of the nucleotides, andmonitoring the reaction products, the identity of the added base isdetermined.

The release of pyrophosphate (PPi) during the DNA polymerase reactioncan be quantitatively measured by many different methods and a number ofenzymatic methods have been described; see Reeves et al., Anal. Biochem.28:282 (1969); Guillory et al., Anal. Biochem. 39:170 (1971); Johnson etal., Anal. Biochem. 15:273 (1968); Cook et al., Anal. Biochem. 91:557(1978); Drake et al., Anal. Biochem. 94:117 (1979); WO93/23564; WO98/28440; WO98/13523; Nyren et al., Anal. Biochem. 151:504 (1985); allof which are incorporated by reference. The latter method allowscontinuous monitoring of PPi and has been termed ELIDA (EnzymaticLuminometric Inorganic Pyrophosphate Detection Assay). A preferredembodiment utilizes any method which can result in the generation of anoptical signal, with preferred embodiments utilizing the generation of achemiluminescent or fluorescent signal.

A preferred method monitors the creation of PPi by the conversion of PPito ATP by the enzyme sulfurylase, and the subsequent production ofvisible light by firefly luciferase (see Ronaghi et al., Science 281:363(1998), incorporated by reference). In this method, the fourdeoxynucleotides (dATP, dGTP, dCTP and dTTP; collectively dNTPs) areadded stepwise to a partial duplex comprising a sequencing primerhybridized to a single stranded DNA template and incubated with DNApolymerase, ATP sulfurylase, luciferase, and optionally anucleotide-degrading enzyme such as apyrase. A dNTP is only incorporatedinto the growing DNA strand if it is complementary to the base in thetemplate strand. The synthesis of DNA is accompanied by the release ofPPi equal in molarity to the incorporated dNTP. The PPi is converted toATP and the light generated by the luciferase is directly proportionalto the amount of ATP. In some cases the unincorporated dNTPs and theproduced ATP are degraded between each cycle by the nucleotide degradingenzyme.

Accordingly, a preferred embodiment of the methods of the invention isas follows. A substrate comprising microspheres containing the targetsequences and extension primers, forming hybridization complexes, isdipped or contacted with a reaction chamber or well comprising a singletype of dNTP, an extension enzyme, and the reagents and enzymesnecessary to detect PPi. If the dNTP is complementary to the base of thetarget portion of the target sequence adjacent to the extension primer,the dNTP is added, releasing PPi and generating detectable light in aregion or zone around the microsphere, which is detected as generallydescribed in U.S. Ser. Nos. 09/151,877 and 09/189,543, and PCTUS98/09163, all of which are hereby incorporated by reference. If thedNTP is not complementary, no detectable signal results. The substrateis then contacted with a second reaction chamber comprising a differentdNTP and the additional components of the assay. This process isrepeated if the identity of a base at a second detection position isdesirable.

In a preferred embodiment, one or more internal control sequences areused. That is, at least one microsphere in the array comprises a knownsequence that can be used to verify that the reactions are proceedingcorrectly. In a preferred embodiment, at least four control sequencesare used, each of which has a different nucleotide at each position: thefirst control sequence will have an adenosine at position 1, the secondwill have a cytosine, the third a guanosine, and the fourth a thymidine,thus ensuring that at least one control sequence is “lighting up” ateach step to serve as an internal control.

The pyrosequencing systems may be configured in a variety of ways; forexample, the target sequence may be attached to the bead in a variety ofways, including direct attachment of the target sequence; the use of acapture probe with a separate extension probe; the use of a captureextender probe, a capture probe and a separate extension probe; the useof adapter sequences in the target sequence with capture and extensionprobes; and the use of a capture probe that also serves as the extensionprobe.

One additional benefit of pyrosequencing for genotyping purposes is thatsince the reaction does not rely on the incorporation of labels into agrowing chain, the unreacted extension primers need not be removed.

Moreover, when the secondary enzymes required to generate the signal areattached to the microspheres, an increased concentration of the requiredenzymes is obtained in the immediate vicinity of the reaction on themicrospheres. This allows for the use of less enzyme and results infaster reaction rates for detection. Thus, preferred embodiments utilizethe attachment, preferably covalently (although as will be appreciatedby those in the art, other attachment mechanisms may be used), of thesecondary enzymes used to generate the signal.

As outlined above for pyrosequencing, the generation and detection ofPPi results in a signal in a zone around a discrete site on an array.Accordingly, in one embodiment, the method of the invention finds use indetecting any enzyme that produces PPi. Enzymes that produce PPiinclude, but are not limited to nucleotide cyclases, nucleotidepolymerases and the like.

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.All references cited herein are incorporated by reference in theirentirety.

EXAMPLES Example 1 Enzyme-Based Sensor Subpopulation A

Bioactive agent: Alkaline phosphatase

Target substrate: fluorescein diphosphate (FDP)

Reported dye ratio: 1:1 ratio of DilC:TRC, where DilC is1,1′,3,3,3′,3′-hexamethyl-indodicarbocyanine iodide and TRC is Texas Redcadaverine

A range of ratios of light intensities are selected that arerepresentative of the optical signature for the dye ratio of thesubpopulation based on the quantum yield of the two dyes. The opticalsignature for this subpopulation is:

ilC λ intensity-ave.DilC background=0.847±0.23

TRC λ intensity-ave.TRC background

Subpopulation B

Bioactive agent: B-Galactosidase;

Target substrate=fluorescein di-B-galactopyranoside (FDG)

Reporter dye ratio: 10:1 ratio of DilC:TRC which translates to anoptical signature of:

DilC λ intensity-ave.DilC background=4.456±1.27

TRC λ intensity-ave.TRC background

Subpopulation C

Bioactive agent: B-glucuronidase

Target substrate=fluorescein di-B-D-glucuronide (FDGicu).

Reporter dye ratio: 1:10 ratio of DilC:TRC, which translates to anoptical signature of:

DilC A intensity-ave. DilC background=0.2136+0.03

TRC A intensity-ave. TRC background

When the microsphere populations are in the presence of one or more ofthe substrates, the respective enzymes on the microspheres catalyze thebreakdown of the substrates producing fluorescein which is fluorescent,emitting light at 530 nanometers when excited at 490 nm. The productionof fluorescein localized to particular beads is then monitored. In thisapproach, the localization of fluorescein around the microspheres isincreased by using a substrate solution of 90% glycerol and 10%substrate. The glycerol inhibits the generated fluorescein fromdiffusing away from the microsphere reaction sites.

During the experiment, images in the encoded wavelengths are firsttaken. Since both DilC and TRC are excited at 577 nm. Each microsphere'semissions at 670 nm, indicative of the presence of DilC and 610 nmindicative of the presence of TRC were recorded using a 595 nm dichroicand an acquisition time of 5 seconds for the CCD 236. Next, the distalend 212 of the fiber bundle is placed in a buffer and another imagetaken while illuminating the beams with 490 nm light. Emissions in the530 nm fluorescein wavelengths were recorded with a 505 nm dichroic. Inthis case, a CCD acquisition time of one second was used. This processprovides a background normalizing image. The buffer was removed and thefiber allowed to dry to avoid substrate solution dilution.

The substrate solution is then introduced and CCD images acquired every30 seconds to a minute for 30 minutes While illuminating themicrospheres with 490 nm light and collecting emissions in the 530 nmrange. Fiber is then placed back in the buffer solution and anotherbackground image captured. Those beads that generate a signal indicativeof fluorescein production are decoded. Depending in the ratio of theintensity of light from the two reporter dyes, DilC:TRC, the bioactiveagent of the optically active beads may be decoded according to thefollowing table.

0.617-1.08  alkaline phosphatase bead 3.188-5.725 β-galactosidase bead0.183-0.243 β-glucunonidese bead

This process is then repeated for the remaining two substrates.

FIGS. 8A-8C are images generated by the CCD 236 when the beadpopulations are exposed to fluorescein diphosphate. FIG. 8A illustratesthe signals from the alkaline phosphatase microspheres when excited at490 nm and recording emissions at 530 nm, emissions at this wavelengthbeing indicative of fluorescein production. FIG. 8B shows the imagecaptured by the CCD when the microspheres are excited at 577 nm andemissions at 670 nm are recorded. This wavelength is an encodingwavelength indicative of the concentration of DilC on the microspheres.Finally, FIG. 8C shows the image when the microspheres are excited with577 nm light and emissions in the 610 nm range are recorded beingindicative of the concentration of TRC in the microspheres.

In a similar vein, FIGS. 9A and 9B are images when the microspheres areexposed to fluorescein β-d-galactosidase. FIG. 9A shows emissions at 530nm indicative of the fluorescein production; and FIG. 9B shows lightemitted at the 670 nm range indicative of the presence of DilC.

These micrographs, FIGS. 8A-8C and 9A-9B illustrate that fluoresceinproduction around the microspheres may be detected as an opticalsignature change indicative of reactions involving the bioactive agentof the microspheres. The micrographs also illustrate that the opticalsignatures may be decoded to determine the chemical functionalities oneach microsphere.

Immunosensor

Three separate subpopulations of beads were used. In subpopulation A,xrabbit antibodies (Ab) were affixed to the surface of the microspheres;in subpopulation B, xgoat antibodies were affixed to the microspheres;and in subpopulation C, xmouse antibodies were affixed to themicrospheres. These three separate subpopulations were identified usinga DilC:TRC encoding ratio similar to that in the previously describedexperiment.

For the first step of the experiment, images at the encoded wavelengthswere captured using 577 nm excitation and looking for emissions at 610and 670 nm. After this decoding, the fiber was placed in a buffer and animage taken at 530 nm with 490 nm excitation. This provided a backgroundnormalizing signal at the fluorescein emission wavelength. Next, thefiber was placed in rabbit IgG antigen (Ag) which is fluoresceinlabeled. Images were then captured every few minutes at the 530 nmemission wavelength for fluorescein. Figs. IOA and IOB are micrographsshowing the image captured by the CCD prior to and subsequent toexposure to a rabbit antigen, which clearly show reaction of theselected microspheres within the population.

Note, if the fluorescein background from the antigen solution is toohigh to see the antibody-antigen signal, the fiber bundle may be placedin a buffer. This removes the background florescence leaving only theAb-Ag signal.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the spirit and scope of theinvention as defined by the appended claims.

1-29. (canceled)
 30. A method of making an array, said methodcomprising: (a) providing a substrate with a surface comprising aplurality of discrete sites; (b) providing a population of beadscomprising a first subpopulation of beads having nucleic acidsassociated therewith and a second subpopulation of beads having enzymesassociated therewith, said beads of the first subpopulation being adifferent size from the beads of the second subpopulation; and (c)randomly distributing said population of beads on said surface such thatsites of said plurality of discrete site have a single bead from saidfirst subpopulation associated therewith and such that beads from saidsecond subpopulation end up at said sites.
 31. The method of claim 30,wherein said first subpopulation and said second subpopulation aresequentially distributed on said surface.
 32. The method of claim 30further comprising making said first subpopulation of beads by attachinga digest of a prokaryotic genome to said beads.
 33. The method of claim30 further comprising making said subpopulation of beads by attaching adigest of a eukaryotic genome to said beads.
 34. The method of claim 30further comprising making said first subpopulation of beads by attachinga copies of fragments of a prokaryotic genome to said beads.
 35. Themethod of claim 30 further comprising making said first subpopulation ofbeads by attaching copies of fragments of a eukaryotic genome to saidbeads.
 36. The method of claim 30, wherein said enzymes are attached tothe beads of the second subpopulation.
 37. The method of claim 30,wherein sites of said plurality of discrete sites lack a single beadfrom said first population.
 38. The method of claim 30 furthercomprising optically coupling a fiber optic bundle to said substrate.39. The method of claim 38, wherein said plurality of discrete sitescomprises a plurality of wells.
 40. The method of claim 39, whereinwells of said plurality of wells are at a density of at least 100 wellsper 1 mm².
 41. The method of claim 39, wherein wells of said pluralityof wells are at a density of at least 10,000 wells per 1 mm².
 42. Themethod of claim 30, wherein said first subpopulation comprises at least10 beads having copies of the same nucleic acid associated therewith.43. A method of making an array, said method comprising: (a) providing asubstrate comprising a plurality of wells; (b) randomly distributingbeads of a first subpopulation of beads into wells of said plurality ofwells such that the wells contain a single bead from the firstsubpopulation of beads, wherein beads of the first subpopulation ofbeads have nucleic acids associated therewith; and (c) distributingbeads of a second subpopulation of beads into the wells containing asingle bead from the first subpopulation of beads, wherein beads of thesecond subpopulation of beads have enzymes associated therewith, andwherein beads of the second subpopulation of beads are smaller thanbeads of said first population of beads.
 44. The method of claim 43further comprising making said first subpopulation of beads by attachinga digest of a prokaryotic genome to said beads.
 45. The method of claim43 further comprising making said subpopulation of beads by attaching adigest of a eukaryotic genome to said beads.
 46. The method of claim 43further comprising making said first subpopulation of beads by attachinga copies of fragments of a prokaryotic genome to said beads.
 47. Themethod of claim 43 further comprising making said first subpopulation ofbeads by attaching copies of fragments of a eukaryotic genome to saidbeads.
 48. The method of claim 43, wherein said enzymes are attached tothe beads of the second subpopulation.
 49. The method of claim 43,wherein wells of said plurality of wells lack a single bead from saidfirst population.
 50. The method of claim 43 further comprisingoptically coupling a fiber optic bundle to said substrate.
 51. Themethod of claim 43, wherein wells of said plurality of wells are at adensity of at least 100 wells per 1 mm².
 52. The method of claim 43,wherein wells of said plurality of wells are at a density of at least10,000 wells per 1 mm².
 53. The method of claim 43, wherein said firstsubpopulation comprises at least 10 beads having copies of the samenucleic acid associated therewith.